Assays for cancer patient monitoring based on levels of analyte components of the plasminogen activator system in body fluid samples

ABSTRACT

The present invention describes methods of examining, screening over time, and monitoring the outcome of a cancer patient who is undergoing treatment or therapy. More specifically, the invention provides a method of monitoring the progression of disease, or the effectiveness of cancer treatment, in a cancer patient by measuring the levels of one or more analytes of the plasminogen activator (uPA) system, namely, uPA, PAI-1 and the complex of uPA:PAI-1, in a sample taken from the cancer patient, preferably, before treatment, at the start of treatment, and at various time intervals during treatment. An increase or elevation in the levels of one or more of the PA system analytes in the cancer patient compared with the levels one or more of the respective PA system analytes in normal control individuals serves as an indicator of cancer advancement or progression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 12/507,737,filed Jul. 22, 2009, which is a continuation of application Ser. No.10/375,646, filed Feb. 27, 2003, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 60/361,219, filedMar. 1, 2002 and U.S. Provisional Application No. 60/368,358, filed Mar.29, 2002, the contents of all of which are incorporated by reference intheir entireties. Additionally, this application claims priority to U.S.application Ser. No. 11/965,323, filed Dec. 27, 2007, which is acontinuation of application Ser. No. 10/375,646, filed Feb. 27, 2003,the contents of all of which are incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to assays for monitoring or assessing theprogress of cancer patients during a course of disease or diseasetreatment or therapy by determining levels of one or more canceranalytes, i.e., components of the plasminogen activator (PA) system,compared to the levels of one or more of these PA system components innormal control individuals. According to the methods described herein,the determination of increases in the levels of one or more of urokinaseplasminogen activator (uPA), PA inhibitor-1 (PAI-1), or a complex of uPAand PAI-1 (the uPA:PAI-1 complex), compared with the levels of theserespective analytes in normal controls is indicative of poor patientand/or treatment outcome relative to disease status.

BACKGROUND OF THE INVENTION

The plasminogen activator (PA) system involves the serine proteasesplasmin and urokinase plasminogen activator (uPA); the serpinsα₂-antiplasmin, plasminogen activator inhibitor type-1 (PAI-1) andplasminogen activator inhibitor type-2 (PAI-2); and the uPA receptor(uPAR). During the past decade, evidence for the involvement ofcomponents of the PA system in cancer metastasis has increased and it isbelieved that the uPA-mediated pathway of plasminogen activation isactive in the cancer process. (P. A. Andreasen et al., 1997, Int. J.Cancer, 72:1-22).

Proteolytic enzymes, such as those of the PA system, are involved incancer invasion and metastasis by virtue of their ability to invade anddegrade basement membranes and extracellular matrix proteins thatsurround normal tissue (J. H. DeWitte et al., 1999, Br. J. Cancer,79:1190-1198; L. A. Liotta et al., 1982 Cancer Metastasis Rev.,1:277-297; K. Dano et al., 1985, Adv. Cancer Res., 44:139-266; P.Mignatti and D. B. Rifkin, 1993, Physiol. Rev., 73:161-195; and P. A.Andreasen et al., 1997, Ibid.). Immunohistochemical and in situobservations of uPA, plasminogen and PAI-1 distribution inadenocarcinomas show that proteinase degradation of the extracellularmatrix occurs as localized invasive foci (J. Grondahl-Hansen et al.,1991, Am. J. Pathol., 138:111-117; C. Pyke et al., 1991, Proc. ThirdIntl. Workshop on the Molecular and Cellular Biology of PlasminogenActivation: Elsinore, 45; C. Pyke et al., 1991, Cancer Res.,51:4067-4071). In the case of angiogenesis, there is also a functionalinteraction between uPA and PAI-1 (E. Bacharach et al., 1992, Proc.Natl. Acad. Sci. USA, 89:10686-10690).

Urokinase plasminogen activator (uPA) is a 52 kilodalton (kDa) serineprotease that is secreted by cells as an inactive, single-chainprecursor called pro-uPA. Enzymatic cleavage of pro-uPA at lysine 158produces an active heterodimer, called high molecular weight uPA(HMW-uPA), which contains two subunits A and B. When pro-uPA is secretedfrom cells, it binds to uPAR on the cell surface through an EGF-likedomain on the A chain. Subsequent binding of plasmin to uPA can convertpro-uPA into the proteolytically active heterodimer. In turn, active uPArapidly converts the inactive plasmin precursor, plasminogen, intoenzymatically active plasmin, which is directly involved inextracellular matrix degradation, as well as in the activation of otherpro-collagenases, some prometalloproteases and latent growth factors (K.Dano et al., 1985, Ibid.; M. J. Duffy, 1992, Clin. Exp. Metastasis,10:145-155; J. R. Pollanen et al., 1991, Adv. Cancer Res., 57:273-282;L. Ossowski, 1992, Cancer Res., 52:D:6754-6760; P. Mignatti and D. B.Rifkin, 1993, Ibid.; and P. A. Andreasen et al., 1997, Ibid.). Theadditional cleavage of uPA after lysine 135 releases the 17 kDa aminoterminal fragment (ATF), leaving the carboxy-terminal low molecularweight uPA (LMW-uPA, 33 kDa), which retains full catalytic activity. (F.Blasi et al., 1990, Seminars in Cancer Biology, 1:117-126).

Both PAI-1 and PAI-2 bind to the catalytically active B chain of uPA toregulate its enzymatic activity. By forming complexes with uPA bound toUPAR on the cell surface, PAI-1 promotes the clearance of proteolyticactivities from the cell surfaces, as well as the recycling of unbounduPAR back to the cell surface, thereby regulating the overall invasiveand metastatic behavior of cancer cells. (P. A. Andreasen et al., 1997,Ibid. and H. A. Chapman et al., 1997, Curr. Op. Cell Biol., 9:714-724).PAI-1 is a 50 kDa glycoprotein serine protease inhibitor that is theprincipal physiological inhibitor of both forms of the plasminogenactivators PA and tissue plasminogen activator (TPA). PAI-1 is secretedin an active form which spontaneously converts to a latent form (G. Denget al., 1995, Thrombosis and Haemostasis, 74:66-70), but it can bestabilized in the active form by binding to the plasma proteinvitronectin (D. A. Lawrence et al., 1994, J. Biol. Chem.,269:15223-15228). Both tumor cells and capillary endothelial cellsexpress higher levels of PAI-1 than do other cell types (K. Bajou etal., 1998, Nature Medicine, 4:923-928). High levels of PAI-1 are thoughtto protect the tumor stroma from degradation by the high amounts of uPAsecreted by cells. (E. Bacharach et al., 1992, Proc. Natl. Acad. Sci.USA, 89:10686-10690; P. Kristensen et al., 1990, Histochemistry,93:559-566). Elevated levels of PAI-1 may also contribute totumor-induced angiogenesis by protecting the extracellular matrixsurrounding the tumor from proteolytic degradation (C. Pyke et al.,1991, Cancer Res., 51:4067-4071). When active uPA is bound to itsreceptor, the subsequent binding of PAI-1 results in internalization anddegradation of the uPA:uPAR:PAI-1 complex. (M. V. Cubellis et al., 1990,The EMBO J., 9:1079-1085). This down-regulation of uPA decreases theamount of active uPA on the cell surface.

Secreted uPA can originate from several cell types, including tumorcells (G. Markus et al., 1983, Cancer Res., 43:5517-5525), adjacentstromal cells and fibroblasts (C. Pyke et al., 1991, Am. J. Pathol.,138:1059-1067). Early studies of PAI-1 and uPA:PAI-1 complexes inoncogenesis involved the use of tumor lysates and cytosols; it was foundthat PAI-1 levels in tumor lysates had a prognostic correlation inbreast cancer. Tumor levels of PAI-1 were also analyzed in lung cancer,colon cancer and renal cell carcinoma; this inhibitor has become anunlikely prognostic marker in tumor tissue for cancer metastasis. (P. A.Andreasen et al., 1997, Ibid.).

Because the PA system components are intricately involved in the processof cancer and cancer spread in a variety of cancer types, which afflictboth genders, it is a problem in the art to be able to accurately andsensitively screen over time to determine and monitor those individualswho are likely to respond, and/or who are responding to, (or notresponding to), or benefiting from (or not benefiting from), anti-cancertherapy(ies), or combination therapies, particularly, molecularlytargeted therapies to the plasminogen activation system. The presentinvention solves such a problem by providing a sensitive and reliableassay method, preferably an immunoassay, to determine levels of PAsystem analyte components in body fluid samples of cancer patientscompared to the levels of these respective PA system components innormal individuals. In addition, the present invention is advantageousin that it is employed to monitor cancer patients undergoing cancer oranti-neoplastic therapies to treat cancers associated with the activityof PA system components to assist in the determination and examinationof cancer treatment regimens and patient progress and/or outcome duringthe course of disease and/or anti-cancer therapy(ies).

SUMMARY OF THE INVENTION

The present invention provides assays (methods) for the analysis of bodyfluid samples from cancer patients to detect and measure levels of PAsystem analytes, namely, uPA, PAI-1 and the complex of uPA:PAI-1, todetermine whether the levels of one or more of these analytes isincreased in cancer patients compared to their respective levels innormal individuals. The determination of a measurable increase in thelevels of one or more of the PA system analytes in a cancer patient'sbody fluid sample, e.g., a plasma or serum sample, compared to thelevels of these PA system analytes in normal controls, affords a meansof monitoring the patient's disease status, and/or patient response orbenefit to cancer therapy, both conventional anti-cancer andanti-neoplastic disease treatments and therapies, e.g., drugs, hormones,and the like, and treatments and therapies that more particularly targetone or more of the PA system components.

A particular aspect of the present invention provides a monitoringmethod in which plasma or serum levels of uPA, PAI-1, and the uPA:PAI-1complex in patients having a cancer or neoplastic disease, for example,of the colon, prostate, breast (mammary), or lung, are monitored duringthe course of cancer or anti-neoplastic treatment, and preferably priorto, or just at, the start of treatment. The determination of an increasein the plasma or serum levels of one or more of these PA system analytesin the cancer patient compared to the normal levels of one or more ofthe PA system analytes allows the practitioner to be able to evaluatethe patient's disease progression and/or outcome of disease. Forexample, based on the monitoring of a patient's PA system analyte levelsover time compared to normal levels of the analytes, as well as to thepatient's own prior-determined levels, a determination can be made as towhether a treatment regimen should be changed, i.e., to be moreaggressive or less aggressive; to determine if the patient is respondingfavorably to his or her treatment; and/or to determine disease status,such as advanced stage or phase of the cancer, or a remission, reductionor regression of the cancer or neoplastic disease.

Another aspect of the present invention provides normal plasma or serumvalues for uPA, PAI-1 and the uPA:PAI-1 complex in healthy individuals.In accordance with the invention, normal ranges of plasma or serumlevels for uPA, PAI-1 and a complex of uPA:PAI-1 have been establishedfor normal males, normal females and both normal males and normalfemales. (Tables 1-3). Normal levels of uPA in serum are provided;normal levels of PAI-1 and the uPA:PAI-1 complex in plasma are provided.

In yet other aspects of the present invention, methods of monitoringcancer or neoplastic disease in patients to select those patients whoare most likely to benefit from anti-cancer or anti-neoplastic diseasetherapies, including those therapies that specifically target componentsof the PA system, are provided and performed as described herein.According to the invention, the methods involve the monitoring of theindividual PA system components, namely, uPA, PAI-1, and the uPA:PAI-1complex, either alone, or in combination with each other, to provideindividualized assessment of patient status before, during and aftercancer treatment; and specialized cancer treatment for the cancerpatient, based on a determination of the levels of one or more of the PAsystem components relative to the levels of these components in normalcontrols.

Further aspects, features and advantages of the present invention willbe better understood upon a reading of the detailed description of theinvention when considered in connection with the accompanyingfigures/drawings.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the PAI-1 levels in normal human plasma. For operator1, the PAI-1 level in normal female plasma was determined to be 33.29ng/ml and the PAI-1 I level in normal male plasma was determined to be23.82 ng/ml; for operator 2, the PAI-1 level in normal female plasma wasdetermined to be 30.13 ng/ml and the PAI-1 I level in normal male plasmawas determined to be 23.18 ng/ml; for operator 3, the PAI-1 level innormal female plasma was determined to be 30.12 ng/ml and the PAI-1 Ilevel in normal male plasma was determined to be 23.46 ng/ml.

FIG. 2 shows the increases in plasma PAI-1 levels by cancer type, i.e.,the percent of patients having plasma PAI-1 levels above the normalcutoff, in patients having breast, colon, lung, or prostate cancers. Inthis analysis, for breast cancer patients, plasma PAI-1 levels in 24% ofthe patients were found to be above plasma PAI-1 levels in normalcontrols; for colon cancer patients, plasma PAI-1 levels in 30% of thepatients were found to be above normal plasma PAI-1 levels; for lungcancer patients, plasma PAI-1 levels in 40% of the patients were foundto be above normal plasma PAI-1 levels; and for prostate cancerpatients, plasma PAI-1 levels in 12% of the patients were found to beabove normal plasma PAI-1 levels.

FIG. 3 illustrates the mean uPA:PAI-1 complex levels in normal male andnormal female plasma.

FIG. 4 shows the increases in plasma uPA:PAI-1 complex levels by cancertype, i.e., the percent of patients having plasma uPA:PAI-1 complexlevels above the normal cutoff, in patients having breast, colon, lungand prostate cancers. In this analysis, for breast cancer patients,plasma uPA:PAI-1 complex levels in 52% of the patients were found to beabove the plasma uPA:PAI-1 complex levels in normal controls; for coloncancer patients, plasma uPA:PAI-1 complex levels in 16% of the patientswere found to be above normal plasma uPA:PAI-1 complex levels; for lungcancer patients, plasma uPA:PAI-1 complex levels in 16% of the patientswere found to be above normal plasma uPA:PAI-1 complex levels; and forprostate cancer patients, plasma uPA:PAI-1 complex levels in 20% of thepatients were found to be above normal plasma uPA:PAI-1 complex levels.

FIG. 5 depicts a graph showing time to progression (TTP) versusprobability of disease progression based on the determination ofelevated serum uPA levels in breast cancer patients. (Example 4).

FIG. 6 depicts a graph showing survival time of a cancer patient as afunction of probability of survival based on the determination ofelevated serum uPA levels in breast cancer patients. (Example 4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an accurate and sensitive method ofexamining cancer or neoplastic disease status, as well as screening overtime and monitoring, cancer patients by measuring the levels of one ormore of the PA system analytes uPA, PAI-1, and the uPA:PAI-1 complex ina patient's body fluid sample to determine if the patient has increasedlevels of these PA system analytes relative or compared to the levels ofthese analytes in normal individuals. The determination of higher orincreased plasma or serum levels of one or more of the uPA, PAI-1, oruPA:PAI-1 complex analytes in a sample from a cancer patient comparedwith the levels of one or more of these analytes in normal individuals,(i.e., a comparison with a normal range, a normal value, or a normalcutoff value), informs the patient, physician, and/or clinician as toone or more of the following parameters: the status of the patient'sdisease state; the response of the patient to cancer or anti-neoplastictreatment or therapy; the benefit or efficacy of a treatment or therapyfor the patient; the progress of therapy, or lack thereof; and/or theclinical course and/or outcome of the disease.

Preferably, according to this invention, the uPA analyte is determinedin a body fluid sample from a human patient; non-limiting examples ofsuch samples include pleural fluid samples, pulmonary or bronchiallavage fluid samples, synovial fluid samples, peritoneal fluid samples,bone marrow aspirate samples, lymph, cerebrospinal fluid, ascites fluidsamples, amniotic fluid samples, sputum samples, bladder washes, semen,urine, saliva, tears, blood, and its components serum and plasma, andthe like. Serum is a preferred body fluid sample for uPA determination,as serum allows for real-time assessment of the plasminogen activationstatus of a cancer patient, allows for repeated testing for patientmonitoring and can be performed in a standardized and quantitativemanner.

Also in accordance with this invention, the PAI-1 and the uPA:PAI-1complex analytes are optimally determined using plasma samples.Therefore, it is to be understood that in the methods described herein,a plasma sample is optimally used for the determination/measurement ofboth PAI-1 and the uPA:PAI-1 complex analytes, while serum, or anotherbody fluid sample, is suitable for the determination/measurement of theuPA analyte. For analyses of uPA according to the present invention,plasma and serum levels of the uPA analyte in the same sample areequivalent. For PAI-1 and uPA:PAI-1 complex analyses according to thepresent invention, plasma is used as a valid sample type. It is also tobe understood that body fluid samples from other mammals, e.g.,non-human primates, and other large and small animals, are also able tobe assayed and monitored by the methods as described herein.

In accordance with the present invention, a patient whose sample is tobe monitored or assessed can be undergoing, or ready to undergo,conventional cancer therapy and/or more unconventional anti-PA systemtherapies, such as those described herein. The present methods alsoencompass a method of determining patient outcome or disease severity,based on the finding of increased plasma or serum levels of the PAsystem analytes in cancer patients compared to the plasma levels ofthese analytes in normal individuals. The methods are particularlyuseful in light of the advent of more unconventional cancer therapies,e.g., small molecule inhibitors of PA system components, that arespecifically targeted to the plasminogen activation system.

As one nonlimiting example, a urokinase/plasmin inhibitor WX-UK1 (asynthetic amidino phenylalanine-type serine protease inhibitor drug),either used alone or in combination with epirubicin (ananthracycline-type cytostatic commonly used in the treatment of humanmammary cancers), has been shown to have anti-tumor and anti-metastaticactivity. (Wilex Biotechnology, Munich, Germany; B. Setyono-Han et al.,2001, Proc. Am. Assoc. Cancer Res., 42:69 (Abstract #371), from the 2001Meeting of the AACR). As another example, not meant to be limiting,other small molecule inhibitors were found to inhibit components of thePA system. One such molecule, a peptide-based uPA receptor antagonist(WX-360), was found to intervene with the uPA/uPAR processes requiredfor tumor angiogenesis; a second molecule, WX-UK1, a synthetic serineprotease inhibitor based on 3-amidino-phenylalanine, was found to reducethe growth rate of primary tumors and the number of metastatic foci atdistant sites in syngeneic rat models of mammary and pancreatic cancers;a third molecule, WX-293 was shown to be a highly selective inhibitor ofuPA. (Wilex Biotechnology, Munich, Germany; J. C. Probst et al., 2001,Proc. Am. Assoc. Cancer Res., 42:69 (Abstract #370), from the 2001Meeting of the AACR).

The present invention encompasses the use of the described in vitroanalysis methods to assess uPA, PAI-1 and uPA:PAI-1 complex levels inpatients having a variety of cancers or neoplastic diseases,particularly those cancers or neoplastic diseases that are associatedwith the abnormal expression or activity of one or more components ofthe PA system. Non-limiting examples of cancers and neoplastic diseasesembraced by this invention include solid tumor cancers, and cancers ofthe skin, lung, trachea, breast (mammary), prostate, gynecologicalcancers, such as those of the cervix, ovary, vulva, vagina andendometrium, urinary tract cancers, such as those of the bladder; andcancers of the pancreas, gall bladder, thyroid, esophagus, head andneck, brain, kidney, liver, stomach (gastric), gastrointestinal, rectumand colon (colorectal).

According to this invention, the PA system analytes uPA, PAI-1 and theuPA:PAI-1 complex can be measured in a sample, e.g., a plasma or serumsample depending upon the analyte, using assays that specifically detectthese components, for example, radioisotopic immunoassays ornon-isotopic immunoassays, e.g., fluorescent immunoassays and enzymaticimmunoassays, such as an enzyme linked immunoassay (ELISA), as arecommercially available, known and practiced in the art. (See, e.g., U.S.Pat. No. 5,422,245 to Lars S, Nielsen et al.; U.S. Patent ApplicationNo. 20020012950 A1 of Lars S, Nielsen et al.; uPA Microtiter ELISA(Bayer Diagnostics/Oncogene Science, Cambridge, Mass.); PAI-1 MicrotiterMicrotiter ELISA (Bayer Diagnostics/Oncogene Science, Cambridge, Mass.);Human uPA:PAI-1 Complex Quantitative ELISA (Bayer Diagnostics/OncogeneScience, Cambridge, Mass.); and uPA, PAI-1, and uPA:PAI-1 ELISA assays(American Diagnostica, Greenwich, Conn.). By way of example, other meansfor determining and measuring the levels of PA system analytes in asample include affinity chromatography, ligand binding assays and lectinbinding assays. Immunoassays, especially non-radioisotopic enzymaticimmunoassays, are preferred. Normal range and normal mean values can bedetermined for the assay being carried out, as is known and practiced inthe art, based on normal (healthy) population samples.

Antibodies directed against the PA system analytes, or antigenic orimmunogenic epitopes thereof, i.e., uPA, PAI-1, and the uPA:PAI-1complex, can be, for example, polyclonal or monoclonal antibodies.Antibodies suitable for use in the assays of the present invention alsoinclude chimeric, single chain, and humanized antibodies, as well asFab, F(ab′)₂, or Fv fragments, or the product of a phage displaylibrary, e.g., an Fab expression library. Various procedures known inthe art may be used for the production of such antibodies and antibodyfragments. Examples of phage display methods that can be used to makeantibodies for use in the present invention include those disclosed inBrinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ames et al.,1995, J. Immunol. Methods, 184:177-186; Kettleborough et al., 1994, Eur.J. Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton etal., 1994, Advances in Immunology, 57:191-280; and in U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108.

Antibodies generated against the PA system analytes can be obtained bydirect injection of an immunogenic uPA, PAI-1, or uPA:PAI-1 preparationinto an animal, or by administering all, or a portion, of the analytepolypeptide to an animal, preferably a nonhuman animal. For thepreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunol. Today, 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producesingle chain antibodies to the uPA, PAI-1, and/or uPA:PAI-1 PA systemanalytes. Also, transgenic mice may be used to express humanizedantibodies to immunogenic uPA, PAI-1, or uPA:PAI-1 complex.

Methods for producing and screening for anti-PA system analyte specificantibodies using hybridoma technology are routine and well known in theart. In a nonlimiting example, mice can be immunized with an immunogen,i.e., uPA, PAI-1, or uPA:PAI-1 complex polypeptide or peptide thereof,or with a cell expressing the polypeptide or peptide. Once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the sera of immunized mice, the spleen is harvested andsplenocytes are isolated. The splenocytes are then fused by well knowntechniques to any suitable myeloma cells, for example cells from cellline SP2/0 or P3X63-AG8.653 available from the ATCC. Hybridomas areselected and cloned by limiting dilution techniques. The hybridomaclones are then assayed by methods known in the art to determine andselect those cells that secrete antibodies capable of binding to apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by injecting mice withpositive hybridoma clones.

PA system analyte polypeptides comprising one or more immunogenicepitopes of the PA system analytes which elicit an antibody response canbe introduced together with a carrier protein, such as an albumin, to ahost animal (such as rabbit, mouse, rat, sheep, or goat). Alternatively,if the polypeptide is of sufficient length (e.g., at least about 25amino acids), the polypeptide can be presented without a carrier.However, immunogenic epitopes comprising as few as 5 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

The PA system analytes, or peptides thereof, can be used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods. See, e.g., Sutcliffe et al., 1983, Science,219:660-666; Wilson et al., 1984, Cell, 37:767-778; and Bittle et al.,1985, J. Gen. Virol., 66:2347-2354). If in vivo immunization is used,animals can be immunized with free peptide; however, the anti-peptideantibody titer may be boosted by coupling the peptide to amacromolecular carrier, such as keyhole limpet hemacyanin (KLH), ortetanus toxoid (TT). For instance, peptides containing cysteine residuescan be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent, such asglutaraldehyde.

Antibodies specific for the PA system analytes can be produced by anymethod known in the art for the synthesis of antibodies, in particular,by chemical synthesis, by intracellular immunization (i.e., intrabodytechnology), or by recombinant expression techniques. Methods ofproducing antibodies include, but are not limited to, hybridomatechnology, EBV transformation, as well as through the use recombinantDNA technology. Recombinant expression of an antibody, or a fragment,derivative, variant or analog thereof, (e.g., a heavy or light chain ofan anti-PA system analyte antibody), requires construction of anexpression vector containing a polynucleotide that encodes the antibody.Once a polynucleotide encoding an antibody molecule or a heavy or lightchain of an antibody, or portion thereof (preferably containing theheavy or light chain variable domain) has been obtained, the vector forthe production of the antibody molecule can be produced by recombinantDNA technology using techniques well known in the art. In vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination methods, which are well known to those skilled in the art,can be used to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. Such vectors can include the nucleotide sequence encoding theconstant region of the antibody molecule (see, e.g., PCT Publication WO86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) andthe variable region of the antibody cloned into such a vector forexpression of the entire heavy or light chain.

The expression vector is then introduced into a host cell byconventional techniques and the transfected cells are cultured byconventional techniques to produce an anti-PA system analyte antibody. Avariety of host expression vector systems can be utilized to express theantibody molecules. Such expression systems represent vehicles by whichthe coding sequences of interest can be expressed, their encodedproducts produced and subsequently purified. These systems alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. Cell expression systems include, but are notlimited, to microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces or Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) or tobacco mosaic virus (TMV)), transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3, NSO cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as E. coli, and more preferably, eukaryotic cells,especially for the expression of whole recombinant antibody molecules,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese hamster ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus, is an effective expressionsystem for antibody production (Foecking et al., 1986, Gene, 45:101;Cockett et al., 1990, BioTechnology, 8:2).

Once an anti-PA system analyte antibody has been produced by an animal,chemically synthesized, or recombinantly expressed, it can be purifiedby any method known in the art for the purification of an immunoglobulinor polypeptide molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigen,Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Typically, an ELISA assay initially involves preparing an antibodyspecific to a given PA system analyte, preferably a monoclonal antibody.In addition, a reporter antibody is used. In some ELISA protocols, thereporter antibody recognizes and binds to the anti-PA analyte-specificmonoclonal antibody. To the reporter antibody is attached a detectablereagent such as a radioactive isotope, a fluorescent moiety, achemiluminescent moiety, or, in an ELISA, an enzyme, such as horseradishperoxidase or alkaline phosphatase.

As is appreciated by those skilled in the art, ELISAs can be performedin a number of assay formats. In one ELISA format, a host sample, e.g.,a patient body fluid sample, is incubated on a solid support, e.g., thewells of a microtiter plate, or a polystyrene dish, to which theproteins in the sample can bind. Any free protein binding sites on thedish are then blocked by incubating with a non-specific protein such asbovine serum albumin. The monoclonal antibody is then added to the solidsupport, e.g., the wells or the dish, and allowed to incubate. Duringthe incubation time, the monoclonal antibodies attach to any PA systemanalyte polypeptides that have attached to the polystyrene dish. Allunbound monoclonal antibody is washed away using an appropriate buffersolution. The reporter antibody, e.g., linked to horseradish peroxidase,is added to the support, thereby resulting in the binding of thereporter antibody to any monoclonal antibody which has bound to the PAsystem analyte present in the sample. Unattached reporter antibody isthen washed away. Peroxidase substrate is added to the support and theamount of color developed in a given time period provides a measurementof the amount of PA system analyte that is present in a given volume ofpatient sample when compared to a standard curve.

In another ELISA format, as described further below and exemplifiedherein, antibody specific for a particular analyte is attached to thesolid support, i.e., the wells of a microtiter plate or a polystyrenedish, and a sample containing analyte is added to the substrate.Detectable reporter antibodies, which bind to the analyte that has boundto the capture antibodies on the support, are then added, after theappropriate incubations and washings, and analyte-antibody complexes aredetected and quantified.

In preferred embodiments, the methods of the present invention involve asandwich ELISA typically performed using microtiter plates. In aparticular embodiment for uPA determination (see, e.g., Examples 1 and4), the microtiter ELISA employs two monoclonal antibodies to human uPAas the capture reagents. Briefly, the capture antibodies are immobilizedon the interior surface of the wells of the microtiter plate. To performthe test, an appropriate volume of serum sample is incubated in thecoated wells to allow binding of the antigen, i.e., uPA, in the sampleby the capture antibodies. The immobilized antigen is then reacted withuPA detector rabbit antiserum. The amount of detector antibody bound tothe antigen is measured by binding it with a goat-anti-rabbit IgGhorseradish peroxidase (HRP) conjugate. Color development by incubationwith o-phenylenediamine (OPD) substrate enables the quantification ofcaptured uPA. The colored reaction product is quantified byspectrophotometry and reflects the amount of uPA analyte that is presentin the plasma sample. A significant change in absorbance indicates apositive result.

In a particular embodiment for PAI-1 determination (see, e.g., Examples2 and 5), the microtiter ELISA employs a monoclonal antibody directedagainst human PAI-1 as the capture reagent. Briefly, the captureantibody is immobilized on the interior surface of the wells of themicrotiter plate. To perform the test, an appropriate volume of sample,e.g., plasma, is incubated in the coated well to allow binding of theantigen by the capture antibody. The immobilized antigen is then reactedwith PAI-1 detector rabbit antiserum. The amount of detector antibodybound to the antigen is measured by binding it with a goat-anti-rabbitIgG horseradish peroxidase (HRP) conjugate. Color development byincubation with o-phenylenediamine (OPD) substrate enables thequantification of captured PAI-1. The colored reaction product isquantified by spectrophotometry and reflects the amount of PAI-1 analytethat is present in the plasma sample.

In a particular embodiment for the determination of uPA:PAI-1 complex(see, e.g., Examples 3 and 5), the microtiter ELISA employs twomonoclonal antibodies to human uPA as the capture reagents. Briefly, thecapture antibodies are immobilized on the interior surface of the wellsof the microtiter plate. To perform the test, an appropriate volume ofsample, e.g., plasma, is incubated in the coated wells to allow bindingof the antigen by the capture antibodies. The immobilized antigen isthen reacted with PAI-1 detector rabbit antiserum. The amount ofdetector antibody bound to the antigen is measured by binding it with agoat-anti-rabbit IgG alkaline phosphatase conjugate. A substratesolution and an amplifier solution are then added, and once completed,the reaction is quenched by the addition of stop solution. The coloredreaction product is quantified by spectrophotometry and reflects theamount of the uPA:PAI-1 protein complex analyte that is present in theplasma sample.

The production of polyclonal and monoclonal antibodies, particularlymonoclonal antibodies, that are specific for uPA, PAI-1, or theuPA:PAI-1 complex is performed using techniques and protocols that areconventionally known and practiced in the art, such as described herein,as well as by example in Lars. S, Nielsen, supra. Also, antibodiesrecognizing the PA system components are commercially available in kitformat. (Bayer Diagnostics/Oncogene Science, Cambridge, Mass.; andAmerican Diagnostica, Greenwich, Conn.).

Standards are used to allow accurate, quantitative determinations of thePA system analytes in the samples undergoing analysis. A microtiterplate reader simultaneously measures the absorbance of the coloredproduct in the standard and the sample wells. Correlating the absorbancevalues of samples with the standards allows the, determination of thelevels of uPA, PAI-1, or the uPA:PAI-1 complex in the sample. Samplesmay be assigned a quantitative value of uPA, PAI-1, or uPA:PAI-1complex, i.e., in picograms per milliliter (pg/ml), or in nanograms permilliliter (ng/ml), of serum or plasma sample.

According to the present invention, a method is provided that permitsthe assessment and/or monitoring of patients who will be likely tobenefit from both traditional and non-traditional treatments andtherapies for cancers and neoplastic disease, particularly those cancersand neoplastic diseases that are associated with the activity of thecomponents of the PA system. The present invention thus embracestesting, screening and monitoring of patients undergoing anti-cancer oranti-neoplastic treatments and therapies, for example, those therapiesinvolving small molecule inhibitors of uPA, PAI-1, or the uPA:PAI-1complex; anti-uPA, anti-PAI-1, or anti-uPA:PAI-1 antibody-basedimmunotherapies, used alone, in combination with each other, and/or incombination with anti-cancer drugs, anti-neoplastic agents,chemotherapeutics and/or radiation and/or surgery, to treat cancerpatients.

An advantage of the present invention is the ability to monitor, orscreen over time, those patients who can benefit from one, or several,of the available cancer therapies, and preferably, to monitor patientsreceiving a particular type of therapy, or a combination therapy, overtime to determine how the patient is faring from the treatment(s), if achange, alteration, or cessation of treatment is warranted; if thepatient's disease has been reduced, ameliorated, or lessened; or if thepatient's disease state or stage has progressed, or become metastatic orinvasive. The cancer treatments embraced herein also include surgeriesto remove or reduce in size a tumor, or tumor burden, in a patient.Accordingly, the methods of the invention are useful to monitor patientprogress and disease status post-surgery.

The identification of the correct patients for a cancer therapyaccording to this invention can provide an increase in the efficacy ofthe treatment and can avoid subjecting a patient to unwanted andlife-threatening side effects of the therapy. By the same token, theability to monitor a patient undergoing a course of therapy using themethods of the present invention can determine whether a patient isadequately responding to therapy over time, to determine if dosage oramount or mode of delivery should be altered or adjusted, and toascertain if a patient is improving during therapy, or is regressing oris entering a more severe or advanced stage of disease, includinginvasion or metastasis, as discussed further herein.

A method of monitoring according to this invention reflects the serial,or sequential, testing or analysis of a cancer patient by testing oranalyzing the patient's body fluid sample over a period of time, such asduring the course of treatment or therapy, or during the course of thepatient's disease. For instance, in serial testing, the same patientprovides a body fluid sample, e.g., serum or plasma, or has sampletaken, for the purpose of observing, checking, or examining the levelsof uPA, PAI-1, or the complex of uPA:PAI-1 in the patient by measuringthe levels of one or more of these analytes during the course oftreatment, and/or during the course of the disease, according to themethods of the invention.

Similarly, a patient can be screened over time to assess the levels ofone or more of the PA system analytes in a body fluid sample for thepurposes of determining the status of his or her disease and/or theefficacy, reaction, and response to cancer or neoplastic diseasetreatments or therapies that he or she is undergoing. It will beappreciated that one or more pretreatment sample(s) is/are optimallytaken from a patient prior to a course of treatment or therapy, or atthe start of the treatment or therapy, to assist in the analysis andevaluation of patient progress and/or response at one or more laterpoints in time during the period that the patient is receiving treatmentand undergoing clinical and medical evaluation.

In monitoring a patient's uPA, PAI-1 or uPA:PAI-1 complex levels over aperiod of time, which may be days, weeks, months, and in some cases,years, or various intervals thereof the patient's body fluid sample,e.g., a serum or plasma sample, is collected at intervals, as determinedby the practitioner, such as a physician or clinician, to determine thelevels of one or more of the PA system analytes in the cancer patientcompared to the respective levels of one or more of these analytes innormal individuals over the course or treatment or disease. For example,patient samples can be taken and monitored every month, every twomonths, or combinations of one, two, or three month intervals accordingto the invention. Quarterly, or more frequent monitoring of patientsamples, is advisable.

The levels of the one or more PA system analytes found in the patientare compared with the respective levels of the one or more of these PAsystem analytes in normal individuals, and with the patient's own PAsystem analyte levels, for example, obtained from prior testing periods,to determine treatment or disease progress or outcome. Accordingly, useof the patient's own PA system analyte levels monitored over time canprovide, for comparison purposes, the patient's own values as aninternal personal control for long-term monitoring of uPA, PAI-1, and/oruPA:PAI-1 complex levels. As described herein, following a course oftreatment or disease, the determination of an increase in one or more ofthe PA system component levels in the cancer patient over time comparedto the respective levels of one or more of these analytes in normalindividuals reflects the ability to determine the severity or stage of apatient's cancer, or the progress, or lack thereof, in the course oroutcome of a patient's cancer therapy or treatment.

For a variety of cancers, e.g., lung cancer, prostate cancer, ovariancancer, gastric cancer, colon cancer and breast (mammary) cancer, anincrease or elevation in the levels of one or more of uPA, PAI-1, or theuPA:PAI-1 complex analytes in a cancer patient's body fluid sample,e.g., serum or plasma, compared to the respective levels of one or moreof these analytes in normal controls following performance of the methodof the invention is indicative of disease progression or severity of thecancer. Elevations or increases in the levels of the PA system analytesin cancer patients is determined by comparing the values obtained fromanalyzing cancer patient samples to the normal control range values. Inperforming the method of the present invention, any value outside of thenormal control range is considered increased or decreased, dependingupon the value. The normal range is the normal mean (i.e., average)value plus/minus (±) two standard deviations.

In monitoring a patient over time, a decrease in the levels of one ormore of a patient's PA system analytes from increased levels compared tonormal range values to levels at or near to the levels of the analytesfound in normal individuals is indicative of treatment progress orefficacy, and/or disease improvement, remission, tumor reduction orelimination, and the like. Likewise, in all of the methods described inthe embodiments of this invention, a determination of a decrease of oneor more of a patient's uPA, PAI-1, or uPA:PAI-1 complex levels from anelevated level to, or approximately to, the respective levels of one ormore of these analytes found in normal individuals provides a furtheraspect of the methods of the invention, in which a patient'simprovement, recovery or remission, and/or treatment progress orefficacy, is able to be ascertained over time following performance ofthe method.

In one embodiment, a cancer patient's sample is analyzed in accordancewith the methods of this invention to determine if there is an increasein the serum or plasma levels of one or more of uPA, PAI-1, or theuPA:PAI-1 complex compared with the respective serum or plasma levels ofone or more of these PA system analytes found to be the normal range ofuPA, PAI-1, and uPA:PAI-1 complex in normal, cancer-free individuals.(Tables 1, 2 and 3). According to the present invention, normal valuesof the PA system analytes in serum or plasma have been determined toallow a reliable and standardized comparison between the levels of theuPA, PAI-1 and the uPA:PAI-1 complex analytes in normal controls and therespective levels of these analytes in cancer patients.

The normal values for the uPA analyte in serum are presented in Table 1in accordance with the present invention:

TABLE 1 Range of Average Normal Normal Values Number Value of uPA in ofuPA in Sample Type Tested Serum Serum* Normal Males n = 34 1064 pg/ml579-1549 pg/ml Normal Females n = 55 1124 pg/ml 469-1778 pg/ml NormalMales + n = 89 1192 pg/ml 459-1924 pg/ml Normal Females *All normalrange and cutoff values represent the mean ± two standard deviations(mean ± 2 SD).

The normal values for the PAI-1 analyte in plasma are presented in Table2 in accordance with the present invention:

TABLE 2 Average Normal Cutoff Normal Number Value of PAI-1 Values ofPAI-1 Sample Type Tested in Plasma in Plasma* Normal Males n = 80 23.52ng/ml >59.38 ng/ml Normal Females n = 80 26.42 ng/ml >66.61 ng/ml NormalMales +  n = 160 25.00 ng/ml >62.71 ng/ml Normal Females *All normalrange and cutoff values represent the mean ± two standard deviations(mean ± 2 SD).

The normal values for the complex of uPA:PAI-1 analyte in plasma arepresented in Table 3 in accordance with the present invention:

TABLE 3 Cutoff Normal Average Normal Values of Value of uPA:PAI-1uPA:PAI-1 Complex in Complex in Sample Type Number Tested Plasma Plasma*Normal Males n = 80 152.57 pg/ml >347.21 pg/ml Normal Females n = 80 79.67 pg/ml >206.63 pg/ml Normal Males +  n = 160 114.97 pg/ml >293.25pg/ml Normal Females *All normal range and cutoff values represent themean ± two standard deviations (mean ± 2 SD).

Another embodiment of the present invention encompasses a method ofmonitoring a cancer patient's course of disease, or the efficacy of acancer patient's treatment or therapy, in which the patient preferablyhas a cancer that involves the activity of PA system components.Preferably, the patient has a cancer selected from cancer of the breast(mammary), colon, bladder, lung and prostate. The patient's treatment ortherapy can involve an anti-plasminogen activation system-specifictreatment or therapy, or more traditional therapies, such as hormonetherapy, chemotherapeutic drug therapy, radiation, or a combination ofany of the foregoing. The method involves measuring levels of one ormore of uPA, PAI-1, or the uPA:PAI-1 complex in a body fluid sample ofthe cancer patient, preferably a serum sample for uPA determination, anda plasma sample for PAI-1 and uPA:PAI-1 complex determination, anddetermining if the levels of one or more of the PA system analytes inthe patient's sample are increased compared to the respective levels ofone or more of these analytes in normal controls during the course ofdisease or cancer treatment. In accordance with the method, an increasein the levels of the PA system analytes in the cancer patient comparedto the respective levels of the PA system analytes in normal controls isindicative of an increase in stage, grade, severity or progression ofthe patient's cancer and/or a lack of efficacy or benefit of the cancertreatment or therapy provided to the patient during a course oftreatment, e.g., poor treatment or clinical outcome. Normal levels,including average normal mean values and normal range values, of each ofthe PA system analytes for comparative purposes in the methods of thepresent invention are set forth in Tables 1, 2 and 3 herein.

As will be understood by the skilled practitioner in the art, themonitoring method according to this invention is preferably, performedin a serial or sequential fashion, using samples taken from a patientduring the course of disease, or a disease treatment regimen, (e.g.,after a number of days, weeks, months, or occasionally, years, orvarious multiples of these intervals) to allow a determination ofdisease progression or outcome, and/or treatment efficacy or outcome. Ifthe sample is amenable to freezing or cold storage, the samples may betaken from a patient (or normal individual) and stored for a period oftime prior to analysis.

In another of its embodiments, the present invention encompasses thedetermination of the amounts or levels of one or more additional cancermarkers in conjunction with the determination of the levels of one ormore of uPA, PAI-1, or the uPA:PAI-1 complex in a sample to be analyzed.Nonlimiting examples of additional cancer markers whose levels aresuitable for monitoring along with the levels of the PA system analytesin a patient sample for particular cancers as described herein includeHER-2/neu, epidermal growth factor receptor (EGFR), complexed PSA(cPSA), p53 autoantibody, the breast cancer marker CA15-3, and the coloncancer marker CA19-9. By combining the determination of the amounts ofone or more of these additional cancer markers with a determination ofthe levels of the PA system analytes compared with the respective normalvalues of these PA system analytes, the ability to determine and monitorthe disease severity of a patient, and/or patient response, and/ordisease outcome can be enhanced and extended.

In a particular aspect of this invention, the above method is performedusing a serum sample from patients having metastatic breast cancer toascertain the levels of uPA in the sera of breast cancer patientscompared to the levels of uPA in the serum of normal individuals. uPAlevels found to be elevated compared to the normal serum level of uPA of1.75 ng/ml or 1750 pg/ml (mean±2 SD), (a normal range of serum uPA of459-1924 pg/ml), were indicative of decreased response to cancertreatment or therapy, a shorter time to progression and a shorteroverall survival time, thereby providing the monitorable status ofpatient disease and/or response to treatment during the course ofdisease or a cancer or neoplastic disease treatment or therapy regimen.(Example 4).

As another particular aspect of this invention, the method is performedusing a plasma sample from patients having various cancers, e.g., colon,prostate, breast, bladder, or lung cancer, to ascertain the levels ofeither or both PAI-1 and the uPA:PAI-1 complex in the plasma of thesecancer patients. (Example 5). For PAI-1, the plasma of patients havingeach of the different types of cancers showed elevated levels of this PAsystem analyte compared with the levels of PAI-1 found in normal plasma.PAI-1 level monitoring can also be indicative of cancer progression,e.g., increased stage or grade of cancer. For colon, breast and lungcancers, the plasma levels of PAI-1 were most elevated in the advancedstage cancer patients, compared to the levels of PAI-1 in normal plasmacontrols, which was determined to have a normal cutoff value of >63ng/ml.

In accordance with the present invention, a determination of the amountof the uPA:PAI-1 complex in plasma can be particularly beneficial, as itcan provide an indication of the amount of active, or actual,proteolysis that may be occurring in conjunction with a patient'scancer, or cancer progression, because uPA and PAI-1 are in their activeforms in the uPA:PAI-1 complex. For colon, prostate, breast and lungcancer patients, as exemplified herein, elevated plasma uPA:PAI-1complex levels compared to the levels of the complex in normal plasmaindicate disease and/or disease severity. For example, the plasma ofpatients having advanced stage colon, breast and lung cancers showedelevated levels of the uPA:PAI-1 complex compared to the levels of thecomplex in normal plasma control serum, which was determined to have anormal cutoff value of >293 pg/ml.

In another embodiment, the present invention provides a method ofmonitoring cancer treatment or the clinical response of a cancer patientundergoing cancer treatment for a cancer, particularly a cancer that isassociated with activities of components of the plasminogen activationsystem. The treatment or therapy can involve targeting of components ofthe plasminogen activation system via small molecules or antibodies.Alternatively, or in addition, the treatment or therapy can involve moretraditional cancer therapies, (e.g., anti-cancer drugs,chemotherapeutics, radiation, hormones), or combinations of any of thetypes of therapies. The method comprises measuring the levels of one ormore of the PA system analytes uPA, PAI-1, or the uPA-PAI-1 complex, ina serum or plasma sample of the cancer patient, preferably before, atthe start of, and during the course of the patient's cancer treatment,and determining if the cancer patient has increased serum or plasmalevels of one or more of the PA system components compared to therespective serum or plasma levels of the PA system components asdetermined for normal individuals for each type of sample analyzed.Serum samples, or other body fluid samples, are preferably monitored foruPA level determination, while plasma samples are preferably monitoredfor the determination of the levels of PAI-1 and the uPA:PAI-1 complex.

The outcome of cancer treatment of the patient is determined based uponthe measurement of increased serum or plasma uPA, PAI-1, and/oruPA:PAI-1 complex levels in the patient compared to the respectivenormal serum or plasma levels of these analytes during the course oftime that the patient is monitored, where increased levels of one ormore of the plasma or serum uPA, PAI-1, or the uPA-PAI-1 complex incancer patients relative to the respective normal levels of theseanalytes in normal serum or plasma correlate with poor treatment orclinical outcome. The method further comprises determining the serum orplasma levels of one or more of uPA, PAI-1, or the uPA:PAI-1 complexafter a patient has undergone treatment for a length of time that isdeemed by the physician or clinician sufficient to allow a showing ofefficacy of the cancer treatment and/or patient response, anddetermining by performance of the method of the invention whether or notthe treated patient's uPA, PAI-1, and/or uPA:PAI-1 complex levels havebeen lowered to, at, or near those of normal individuals. A lowering ordecrease in the patient's uPA, PAI-1, and/or uPA:PAI-1 complex levelsduring or following a course of treatment or therapy relative to therespective levels of these analytes in normal controls indicatesprogress and/or efficacy of the cancer treatment or therapy.

In another of its aspects, the present invention encompasses a method ofmonitoring disease severity or progression of a cancer patient. Themethod comprises measuring levels of one or more of uPA, PAM, or theuPA:PAI-1 complex in a serum or plasma sample of the cancer patient anddetermining if the cancer patient has increased serum or plasma levelsof one or more of the uPA, PAI-1, or uPA:PAI-1 complex analytes comparedto the respective normal cutoff serum or plasma level values of one ormore of these analytes in normal individuals, e.g., males plus females,are provided in Tables 1, 2 and 3 herein. In the method, cancer severityor progression is monitored in the patient based upon elevated orincreased levels in the patient's sample compared to the respectivenormal PA system analyte levels in normal individuals. According to thismethod, the most severe cancer stage correlates with the most elevatedlevels of plasma or serum uPA, PAI, or uPA:PAI-1 complex compared to therespective normal control levels of these PA system analytes. Also inaccordance with this method, the following normal cutoff values areconsidered for comparative purposes in the determination of elevatedserum or plasma levels in cancer patients: (i) normal uPA serum level isin the range of about 459 pg/ml to about 1924 pg/ml, with a normal meanvalue of uPA in male serum of about 1064 pg/ml±2 SD, and a normal meanvalue of uPA in female serum of about 1124 pg/ml±2 SD; (ii) the cutofffor normal PAI-1 plasma level is in the range of >62.71 ng/ml, with anormal mean value of PAI-1 in male plasma of >23.52 ng/ml±2 SD(cutoff>59.38 ng/ml), and a normal mean value of PAI-1 in female plasmaof >24.42 ng/ml±2 SD (cutoff>66.61 ng/ml); and (iii) the cutoff fornormal uPA:PAI-1 complex plasma level is in the range of >293.25 pg/ml,with a normal mean value of the uPA:PAI-1 complex in male plasmaof >152.57 pg/ml 2 SD (cutoff>347.21 pg/ml), and a normal mean value ofthe uPA:PAI-1 complex in female plasma of >79.67 pg/ml±2 SD(cutoff>206.63 pg/ml).

In yet another of its embodiments, the present invention provides amethod of monitoring cancer treatment, or efficacy thereof, in a cancerpatient undergoing such treatment. The method involves measuring theserum or plasma levels of one or more of uPA, PAI-1, or the uPA:PAI-1complex in a cancer patient and determining if the levels of one or moreof these PA system analytes in the patient increases during the cancertreatment compared to the respective levels of one or more of theanalytes determined in the serum or plasma samples of normal controls,where an elevation or increase in one or more of the uPA, PAI-1, oruPA:PAI-1 complex levels in the cancer patient compared to therespective levels of one or more of these PA system analytes in normalcontrol plasma or serum during the monitoring period indicates one ormore of the following: (i) cancer progression; (ii) a more severe stageof the cancer; (iii) lack of response by the patient to the cancertreatment; or (iv) poor outcome or shorter survival time.

In addition, if, during the course of monitoring the levels of one ormore of the PA system analytes in the patient undergoing treatment, achange is found in the levels of one or more of these analytes beingmeasured, such that a decrease in the levels of one or more of the PAsystem analytes is determined, or a decline in the levels of one or moreof the PA system analytes resulting in values at or near the respectivenormal values of one or more of these analytes is determined, afterhaving monitored an increase or elevation in these levels for a periodof time during a patient's treatment, an assessment can be made as toone or more of the following events: (i) the patient is progressing wellon the treatment, (ii) the treatment is effective; (iii) the patient isresponding to the treatment; and/or (iv) the patient's cancer is notprogressing or has been ameliorated or eliminated by the treatment.

In accordance with the present invention, such a method of monitoringand assessment of one or more of the uPA, PAI-1, or uPA:PAI-1 complexlevels during a patient's course of treatment or therapy, compared withthe respective normal level values of one or more of these analytes, canprovide the physician or clinician with a determination of a patient'sprogress, or regression, as the case may be, as a consequence of aparticular cancer or anti-neoplastic disease treatment or therapy. Sucha determination advantageously allows tailoring of the treatment ortherapy to better or more aggressively attack (or treat) a cancer; italso allows altering dosage, mode of administration, modifying theregimen, or combining therapies to achieve a more effective overalltreatment and outcome for the individual patient.

This approach provided by the present invention also allows thepractitioner to determine whether dosage or mode of administrationshould be altered, or whether the drug regimen should be modified, forexample, by combining therapies or discontinuing therapies, to try toachieve a more effective overall treatment and outcome for the patient.As an example, if it is determined by way of practicing the presentinvention that a patient has a high likelihood of relapse (due to themonitoring of a continued increase in the levels of one or more of uPA,PAI-1 and/or the uPA:PAI-1 complex compared with the respective normallevels of these PA system analytes over a number of testing times orintervals), the patient can be treated more rigorously, such as by usingsystemic chemotherapy and/or radiation therapy, or other treatmentcombinations. Similarly, when the levels of one or more of the PA systemanalytes monitored by the present methods are determined to decreaseover time, i.e., to levels at, or close to, those of normal controls,less aggressive therapies can be decided upon. The ability to select apersonalized course of therapy or treatment regimen, i.e., to be able tochoose a less aggressive treatment at or close to the start oftreatment, or to alter treatment from aggressive to less aggressive at atime prior to the conventional end of a treatment regimen on the basisof the monitoring analysis methods of this invention, can provide lessanguish and suffering for the patient on both an emotional and physicallevel.

Another embodiment of the invention encompasses a method of determiningif a cancer patient is a candidate for anti-plasminogen activationsystem cancer therapy. The method comprises measuring the levels ofPAI-1 or uPA:PAI-1 complex in a plasma sample of the cancer patient;determining if the levels of PAI-1 or uPA:PAI-1 complex in the plasma ofthe cancer patient are elevated compared to the respective levels ofPAI-1 or the uPA:PAI-1 complex in the plasma of normal controls; andselecting the patient having elevated plasma PAI-1 or uPA:PAI-complexlevels as a candidate for anti-plasminogen activation system therapy,based on the determination of an elevated level of PAI-1 or uPA:PAI-1complex in the plasma of the cancer patient compared to the plasma levelof PAI-1 or uPA:PAI-1 complex in the normal controls. The levels ofPAI-1 or uPA:PAI-1 complex in normal individuals are presented in Tables2 and 3 herein. As a nonlimiting example according to this method, theanti-plasminogen activator system therapy can be a protease inhibitor,such as amidino phenylalanine-type serine protease inhibitor, or aphenylguanidine-based inhibitor, or a uPA receptor antagonist. Inaddition, the therapy can be administered in combination with at leastone biologically active agent, such as one or more of i) drugs; ii)hormones; or iii) synthetic compounds, such as epirubicin.

The above method can also be performed by measuring the levels of uPA ina cancer patient's body fluid sample, preferably serum, to detect anelevation or increase in the uPA level compared with the normal level ofuPA detected in healthy control individuals. An increase in uPA levelsin the patient is also, or further, indicative of the patient'slikelihood to be a good candidate for treatment or therapy targetingcomponents of the PA system. Accordingly, by the practice of thisinvention, a given patient can be started on a decided upon treatmentregimen, monitored throughout the treatment, and undergo alteration ofthe treatment, or not, depending on the performance of the method inwhich detection of elevated or non-elevated levels of one or more of thePA system analytes compared to the respective levels in normalindividuals determines how a patient's therapy will be most beneficiallyindividualized and controlled over time.

It is to be understood that in all of the embodiments describing themethods according to the present invention, the monitoring of a cancerpatient for disease progression or outcome, or for cancer treatment ortherapy efficacy or outcome, can include the analysis of a pretreatmentsample taken from the patient at a first time point, and can alsoinclude the analysis of a patient's samples taken at a second, third,fourth, or subsequent time, during the course of disease or during acancer or anti-neoplastic treatment or therapy regimen, or a combinationof treatment or therapy regimens.

In another embodiment, the present invention encompasses a method ofmonitoring patient response to cancer therapy or treatment for a patienthaving metastatic cancer, particularly breast cancer or prostate cancer.The method involves measuring one or more of the following: the leveluPA in a body fluid sample of the cancer patient; the level of PAI-1 ina plasma sample of the cancer patient; and/or the level of the uPA:PAI-1complex in a plasma sample of the cancer patient prior to, or at thestart of, cancer therapy or treatment. At various times during thecourse of treatment, the levels of uPA, PAI-1, and/or the uPA:PAI-1complex in the patient's samples are respectively examined to determineif the levels of one or more of these PA system analytes in the cancerpatient is increased, decreased, or the same as the respective levels ofthese same analytes in normal control individuals. If, by practice ofthe method, it is established that the patient having cancer, e.g.,metastatic breast cancer, has increased or elevated uPA, PAI-1, and/oruPA:PAI-1 complex levels compared to the levels of one or more of thesePA system component analytes in normal controls, one or more of thefollowing can be determined: (i) the patient has ongoing or progressingmetastasis; (ii) there is a likelihood of a decreased or poor responseto cancer therapy or treatment; (iii) the patient's survival outcome ispoor. If a finding of decreased levels, or the same levels, of the PAsystem analytes are present in the patient's sample compared with therespective levels of these same analytes in normal control individualsduring the course of monitoring the patient, a more optimisticassessment of patient progress, response to treatment and therapy, andsurvival outcome can be concluded.

An especially heartening determination for the patient and the physicianor clinician is the observation of a change in the patient's uPA, PAI-1,and/or uPA-PAI-1 complex levels during a patient's course of treatment,i.e., from a finding of elevated levels of uPA, PAI-1, and/or theuPA:PAI-1 complex in pretreatment or early treatment patient samples toa finding in the patient of decreased uPA, PAI-1, and/or uPA:PAI-1complex levels, or levels of uPA, PAI-1, and/or uPA:PAI-1 complex thatare at, or close to, the respective levels of the uPA, PAI-1, and/oruPA:PAI-1 complex analytes determined in normal individuals. Such afinding reflects, among other things, patient improvement from thetreatment or therapy, successful treatment outcome, and/or a change to aless serious stage or phase of disease. In the method, for comparativepurposes, the normal range of uPA level is 459-1924 pg/ml (averagenormal cutoff: 1192 pg/ml±2 SD); the normal range of PAI-1 level isgreater than 63 mg/ml (average normal cutoff: 25 ng/ml±2 SD); and thenormal range of the uPA:PAI-1 complex level is greater than 293 pg/ml(average normal cutoff: 115 pg/ml±2 SD). In addition, the method can bepracticed by including a measurement or determination of the levels ofone or more oncoprotein markers selected from the group consisting ofHER-2/neu, epidermal growth factor receptor (EGFR), complexed PSA(cPSA), CA15-3 breast cancer marker, CA19-9 colon cancer marker, and p53autoantibody, which can also assist in the determination of patientoutcome following treatment, or during the course of disease.

In another of its embodiments, the present invention encompasses thedetermination of uPA levels in a body fluid sample, e.g., serum, in apatient having pancreatic cancer to assess whether the levels of this PAsystem analyte are elevated or increased in pancreatic cancer patientscompared to uPA levels in normal control individuals, particularly forthe purposes of monitoring pancreatic cancer disease course and/ortreatment outcome in patients. The finding of an elevation in uPA levelsin body fluid, e.g., serum, samples of both male and female patientscompared with gender-matched healthy controls allows an assessment ofthe status of the pancreatic cancer patient's disease over time, and ofthe progress of a patient undergoing treatment for his or her pancreaticcancer in accordance with the monitoring feature of this invention.(Example 7).

Further, in a manner similar to the other embodiments of this invention,changes in the uPA levels compared with normal levels, and/or comparedwith the patient's own uPA levels, during the course of monitoring thepatient's disease or treatment status, can serve as an indicator of thepancreatic cancer patient's progress, or lack thereof, in diseaseadvancement and/or treatment. More specifically, if, during the courseof monitoring the pancreatic cancer patient, the patient's uPA levelsdecrease so that the uPA levels approximate those of normal controls,such a result can be encouraging by serving to assess a favorable courseof disease, or treatment outcome for the patient. Also, the patient'sown uPA levels can be assessed relative to each other to determinepatient progress and response, or lack thereof, in disease advancementor treatment outcome over time during the monitoring period.

In addition to monitoring the uPA levels in a body fluid sample ofpancreatic cancer patients, the co-measurement and determination of thelevels of one or more oncoprotein marker analytes can assist in thedetermination of patient outcome resulting from disease, or patientoutcome during the course of disease, or during or following the courseof a therapy regimen to treat pancreatic cancer. The co-analysis ofother markers is preferably performed using a serum sample from thepancreatic cancer patient. Such other markers include, withoutlimitation, HER-2/neu, epidermal growth factor receptor (EGFR),complexed PSA (cPSA), CA15-3 breast cancer marker, CA 19-9 colon cancermarker, CEA antigen and p53 autoantibody, with HER-2/neu, CEA and CA19-9markers being preferred. (Example 8). The finding of an increase in oneor more of the cancer markers, other than the PA system analyte markers,such as uPA, in a sample from a pancreatic cancer patient can indicategreater severity of disease, or a poorer disease or treatment outcome.

The invention can be illustrated by the following embodiments enumeratedin the numbered paragraphs below:

1. A method of monitoring cancer progression, or the efficacy of cancertreatment or therapy, in a cancer patient, comprising: (a) measuring thelevels of one or more of the following plasminogen activator (uPA)system components in a body fluid sample from the cancer patient priorto, or at the start of, cancer treatment or therapy: (i) uPA in a bodyfluid sample, (ii) PAI-1 in a plasma sample; and (iii) uPA:PAI-1 complexin a plasma sample; (b) determining if the sample levels of one or moreof the uPA, PAI-1, or uPA:PAI-1 complex in the cancer patient isincreased compared to normal levels of each of the respectiveplasminogen activator system components in normal controls to obtain afirst value for the PA system analytes in the patient; (c) measuring thelevels of one or more of (i) uPA in a body fluid sample of the cancerpatient; (ii) PAI-1 in a plasma sample of the cancer patient; or (iii)the uPA:PAI-1 complex in a plasma sample of the cancer patient duringand/or following a course of cancer treatment or therapy; (d)determining if the patient's sample levels of one or more of the uPA,system components is increased compared to the normal levels of each ofthe respective plasminogen activator system components in normalcontrols during and/or following the course of cancer treatment ortherapy; and (e) establishing that the cancer treatment or therapy is oris not effective; wherein an increase or elevation in the cancerpatient's sample levels of one or more of the uPA, PAI-1 or uPA:PAI-1complex during or following the cancer treatment or therapy comparedwith the levels of one or more of the uPA, PAI-1 or uPA:PAI-1 complex innormal controls and relative to the first value of (a) indicates thatthe cancer treatment or therapy is not effective or that the patient isnot responding to the treatment or therapy; and further wherein adecrease in the sample levels of one or more of the uPA, PAI-1 oruPA:PAI-1 complex during or following the cancer treatment or therapycompared with the levels of one or more of the uPA, PAI-1 or uPA:PAI-1complex in normal controls and compared to the first value of (a)indicates effective treatment or therapy, or a favorable response by thecancer patient.

2. The method according to embodiment 1, wherein the cancer is a solidtumor cancer.

3. The method according to embodiment 2, wherein the solid tumor canceris selected from skin, lung, trachea, breast (mammary), prostate,cervix, ovary, vulva, vagina, endometrium, bladder; pancreas, gallbladder, thyroid, esophagus, head and neck, brain, kidney, liver,stomach, rectum, or colon cancer.

4. The method according to embodiment 1, wherein the cancer is breastcancer, pancreatic cancer, or prostate cancer.

5. The method according to embodiment 1, wherein the cancer patient'ssample uPA level is considered elevated or increased if it is above anormal uPA value of 1924 pg/ml; wherein the cancer patient's samplePAI-1 level is considered elevated or increased if it is above a normalPAI-1 value of 63 ng/ml; and wherein the cancer patient's sampleuPA:PAI-1 complex level is considered elevated or increased if it isabove a normal uPA:PAI-1 complex value of 293 pg/ml.

6. A method of monitoring cancer treatment, or efficacy thereof, in acancer patient undergoing such treatment, comprising: (a) measuringlevels of a complex of plasminogen activator (uPA) and uPA inhibitorPAI-1 (uPA:PAI-1 complex) in a plasma sample of the cancer patient; and(b) determining if the plasma levels of the uPA:PAI-1 complex increaseduring the cancer treatment compared to the plasma levels of theuPA:PAI-1 complex in normal controls; wherein an increase in the plasmalevels of the uPA:PAI-1 complex in the cancer patient compared to theplasma levels of the complex in normal controls during the monitoringperiod indicates one or more of the following: (i) cancer progression,(ii) a more severe stage of the cancer, or (iii) lack of response by thepatient to the cancer treatment.

7. The method according to embodiment 6, wherein the cancer patientshave a solid tumor cancer.

8. The method according to embodiment 6, wherein the cancer patientshave a cancer selected from skin, lung, trachea, breast (mammary),prostate, cervix, ovary, vulva, vagina, endometrium, bladder; pancreas,gall bladder, thyroid, esophagus, head and neck, brain, kidney, liver,stomach, rectum, or colon cancer.

9. The method according to embodiment 6, wherein the normal plasmauPA:PAI-1 complex level is greater than about 293 pg/ml.

10. The method according to embodiment 6, wherein the average normalplasma uPA:PAI-1 complex cutoff level in normal males is greater thanabout 347 pg/ml.

11. The method according to embodiment 6, wherein the normal plasmauPA:PAI-1 complex cutoff level in normal females is greater than about207 pg/ml.

12. The method according to embodiment 6, wherein the plasma uPA:PAI-1complex levels are determined by an enzyme linked immunosorbent assay(ELISA).

13. A method of monitoring cancer treatment, or efficacy thereof, in acancer patient undergoing such treatment, comprising: (a) measuringlevels of a complex of plasminogen activator inhibitor-1 (PAI-1) in aplasma sample of the cancer patient; and (b) determining if the plasmalevels of PAI-1 increase during the cancer treatment compared to theplasma levels of PAI-1 in normal controls; wherein an increase in theplasma levels of PAI-1 in the cancer patient compared with the plasmalevels of PAI-1 in normal controls during the monitoring periodindicates one or more of the following: (i) cancer progression, (ii) amore severe stage of the cancer, or (iii) lack of response by thepatient to the cancer treatment.

14. The method according to embodiment 13, wherein the cancer patientshave a solid tumor cancer.

15. The method according to embodiment 13, wherein the cancer patientshave a cancer selected from skin, lung, trachea, breast (mammary),prostate, cervix, ovary, vulva, vagina, endometrium, bladder; pancreas,gall bladder, thyroid, esophagus, head and neck, brain, kidney, liver,stomach, rectum, or colon cancer.

16. The method according to embodiment 13, wherein the normal plasmaPAI-1 cutoff level is greater than about 63 ng/ml.

17. The method according to embodiment 13, wherein the average normalplasma PAI-1 cutoff level in normal males is greater than about 59ng/ml.

18. The method according to embodiment 13, wherein the normal plasma PAIcutoff level in normal females is greater than about 67 ng/ml.

19. The method according to embodiment 13, wherein the plasma PAI-1levels are determined by an enzyme linked immunosorbent assay (ELISA).

20. A method of monitoring patient response to cancer therapy ortreatment for a patient having metastatic cancer, comprising: (a)measuring levels of plasminogen activator (uPA) in a body fluid sampleof the cancer patient prior to cancer therapy or treatment; (b)determining if the uPA levels of the patient are increased compared tothe uPA levels in normal controls; the normal control range of uPAlevels being 459-1924 pg/ml; and (c) establishing that the metastaticcancer patient having increased uPA levels compared with the uPA levelsof normal controls has one or more of (i) ongoing or progressingmetastasis; (ii) a likelihood of a decreased or poor response to cancertherapy or treatment; and (iii) a shorter survival outcome.

21. The method according to embodiment 20, wherein the body fluid sampleis serum or plasma.

22. The method according to embodiment 20, wherein the body fluid sampleis serum.

23. The method according to embodiment 20, wherein the metastatic canceris a metastatic solid tumor cancer.

24. The method according to embodiment 20, wherein the metastatic canceris selected from the group consisting of metastatic cancer of the skin,lung, trachea, breast (mammary), prostate, cervix, ovary, vulva, vagina,endometrium, bladder; pancreas, gall bladder, thyroid, esophagus, headand neck, brain, kidney, liver, stomach, rectum, or colon cancer.

25. The method according to embodiment 20, wherein the metastatic canceris metastatic breast cancer or metastatic prostate cancer.

26. The method according to embodiment 20, wherein the uPA level isdetermined using an enzyme linked immunosorbent assay (ELISA).

27. The method according to embodiment 20, wherein the uPA levels in thepatient's sample are determined in conjunction with the determination ofthe levels of one or more oncoprotein markers, and further wherein thelevels of the one or more markers are correlated with patient outcome.

28. The method according to embodiment 27, wherein the one or moreoncoprotein markers is selected from the group consisting of HER-2/neu,epidermal growth factor receptor (EGFR), complexed PSA (cPSA), p53autoantibody, the breast cancer marker CA15-3 and the colon cancermarker CA19-9.

29. A method of determining if a cancer patient is a candidate foranti-plasminogen activation system cancer therapy, comprising: (a)measuring the levels of plasminogen activator inhibitor-1 (PAI-1) or acomplex of plasminogen activator and plasminogen activator inhibitor-1(uPA:PAI-1 complex) in a plasma sample of the cancer patient; (b)determining if the levels of PAI-1 or the uPA:PAI-1 complex in theplasma of the cancer patient are elevated compared to the normal rangelevels of PAI-1 or the uPA:PAI-1 complex in the plasma of normalcontrols; and (c) selecting the patient having elevated plasma PAI-1 oruPA-PAI-1 complex levels as a candidate for anti-plasminogen activationsystem therapy, based on the determination of elevated levels of PAI-1or uPA:PAI-1 in the plasma of the cancer patient compared to the normalrange plasma levels of PAI-1 or uPA:PAI-1 complex in the normalcontrols.

30. The method according to embodiment 29, wherein the anti-plasminogenactivator system therapy is selected from serine protease inhibitors oruPA receptor antagonists.

31. The method according to embodiment 30, wherein the serine proteaseinhibitor is an amidino phenylalanine-type serine protease inhibitor.

32. The method according to any one of embodiments 1, 6, 13, 20, or 29,wherein the anti-cancer treatment or therapy is administered incombination with at least one biologically active agent, and furtherwherein the biologically active agent is selected from one or more of i)drugs; ii) hormones; or iii) synthetic compounds.

33. The method according to embodiment 32, wherein the drug isepirubicin.

34. The method according to embodiment 29, wherein the plasma PAI-1levels are determined by an enzyme linked immunosorbent assay (ELISA).

35. The method according to embodiment 29, wherein the normal plasmaPAI-1 cutoff level is greater than about 63 ng/ml, and the normal plasmauPA:PAI-1 complex cutoff level is greater than 293 pg/ml.

36. The method according to embodiment 29, wherein the cancer patienthas a solid tumor cancer.

37. The method according to embodiment 29, wherein the cancer patienthas a cancer selected from breast cancer, colon cancer, lung cancer,ovarian cancer, or prostate cancer.

38. The method according to embodiment 29, wherein the plasma uPA:PAI-1complex levels are determined by an enzyme linked immunosorbent assay(ELISA).

39. The method according to embodiment 29, further wherein plasma levelsof plasminogen activator (uPA) in cancer patients are determined, andwherein an elevated level of uPA in plasma of cancer patients comparedto uPA normal range levels in normal plasma controls indicates furtherlikelihood of benefit of the cancer patient by therapies targetingcomponents of the plasminogen activation system.

40. The method according to embodiment 39, wherein the normal range uPAlevel is 459-1924 pg/ml.

41. A method of monitoring cancer progression, or the efficacy of cancertreatment or therapy, in a cancer patient, comprising: (a) measuring thelevels of one or more of the following plasminogen activator (PA) systemanalytes in a body fluid sample from the cancer patient: (i) uPA analytein a body fluid sample, (ii) PAI-1 analyte in a plasma sample; and (iii)uPA:PAI-1 complex analyte in a plasma sample; and (b) determining if thesample levels of one or more of the uPA, PAI-1, or uPA:PAI-1 complexanalytes in the cancer patient is increased or elevated during thecancer treatment or therapy compared to normal levels of each of therespective plasminogen activator system analytes in normal controls;wherein an increase or elevation in the sample levels of one or more ofthe uPA, PAI-1, or uPA:PAI-1 complex analytes in the cancer patientcompared to the respective normal levels of the analytes during themonitoring period indicates one or more of the following: (i) cancerprogression, (ii) a more severe stage of the cancer, or (iii) lack ofresponse by the patient to the cancer treatment or therapy; and furtherwherein the cancer patient's sample uPA level is considered elevated orincreased if it is above a normal uPA value of 1924 pg/ml; the cancerpatient's sample PAI-1 level is considered elevated or increased if itis above a normal PAI-1 value of 63 ng/ml; and the cancer patient'ssample uPA:PAI-1 complex level is considered elevated or increased if itis above a normal uPA:PAI-1 complex value of 293 pg/ml.

42. The method according to embodiment 41, wherein the cancer patienthas a solid tumor cancer.

43. The method according to embodiment 41, wherein the cancer patienthas a cancer selected from lung, breast (mammary), prostate, cervix,ovary, vulva, vagina, endometrium, bladder, esophagus, head and neck,kidney, liver, stomach, or colon cancer.

44. The method according to embodiment 43, wherein the cancer is breastcancer, ovarian cancer, colon cancer, lung cancer, or prostate cancer.

EXAMPLES

The following examples describe specific aspects of the invention toillustrate the invention and provide a description of the presentmethods for those of skill in the art. The examples should not beconstrued as limiting the invention, as the examples merely providespecific methodology useful in understanding and practice of theinvention and its various aspects.

Example 1 Enzyme Linked Immunoassay (ELISA) to Measure Levels of uPA ina Body Fluid Sample

Serum and plasma samples were centrifuged in a tabletop microcentrifugeto remove all flocculent material and/or particulate matter. Aftercentrifugation, the samples were analyzed without further treatment, orwere stored at −70° C. for future analysis. The initial concentration ofthe serum sample examined did not exceed a concentration of 10% (i.e., a1:10 dilution of sample in sample diluent), (Bayer Diagnostics/OncogeneScience uPA Microtiter ELISA assay kit manual).

The Bayer Diagnostics/Oncogene Science uPA Microtiter ELISA detects 25pg/ml of uPA analyte in a test sample. The signal of the 25 pg/mlstandard is greater than two times the zero (background) signal. Inaddition, the uPA ELISA is specific for the detection of uPA; forexample, no cross-reactivity is detected against tPA and cathepsin D,even at high challenge doses.

Detailed Protocol

The uPA Microtiter ELISA kit (commercially available from BayerDiagnostics/Oncogene Science, Cambridge, Mass.) used in this Example hasremovable strips of wells so that the assay can be performed on multipleoccasions, and only the number of wells needed are used. A standardcurve was included each time that samples were analyzed. The standardcurve performed for each separate assay required 12 wells (6 standardsrun in duplicate). The six standards and the test samples were run induplicate. For greater accuracy, each sample was tested at more than oneconcentration. The contents of the uPA Microtiter ELISA assay manual(Bayer Diagnostics/Oncogene Science, Cambridge, Mass.) are herebyincorporated by reference herein in their entirety.

The uPA standards are housed in six separate vials containinglyophilized pro-uPA. Standards levels were calibrated in mass units(picograms per ml) using an immunoaffinity purified pro-uPA preparation.The standards therefore quantify pro-uPA as well as HMW-uPA (both 52kDa). Low molecular weight uPA (LMW-uPA) is also detected by the assay,but measurement of this form using the standards can overestimate thequantity present. This should not adversely affect most typicalmeasurements of uPA; however, in cases where LMW-uPA is known to bepresent at a significant level, the detection of LMW-uPA should beconsidered. The standards vials were reconstituted with 1 ml of highquality deionized water. As provided, standard 6 contained 350 pg/mlpro-uPA; standard 5 contained pro-uPA at 250 pg/ml; standard 4 containedpro-uPA at 150 pg/ml; standard 3 contained pro-uPA at 75 pg/ml; standard2 contained pro-uPA at 25 pg/ml; and standard 1 contained pro-uPA at 0pg/ml.

The appropriate number of 8-well strips was selected from the microtiterplate; the remaining unused strips were saved for subsequent use. Insome cases, the entire plate was used. A working 1× solution ofplatewash buffer, as provided in the kit (Bayer Diagnostics/OncogeneScience, Cambridge, Mass.) was prepared by adding one part platewashconcentrate to 19 parts of deionized water and mixing well. The totalvolume required depended on the washing method used, e.g., automaticmicroplate washer, manual microplate washer, or hand-held syringe.Approximately 1 liter of the wash buffer was needed to prime anautomated washer and run one microtiter plate. The platewash buffer wasfreshly prepared on the day of use and used at room temperature (18-27°C.).

To perform a same-day assay, samples, standards and other kit reagentswere warmed to room temperature prior to addition to the microtiterplate wells and starting the assay. The wells of the microtiter plateswere precoated with two monoclonal antibodies directed against human uPAas capture reagents. As necessary, samples were diluted in samplediluent containing BSA and 0.1% sodium azide, as provided. (BayerDiagnostics/Oncogene Science uPA ELISA kit). The standards and dilutedsamples were gently mixed without foaming and 100 pl were added induplicate to the wells of the plates. Four wells were set up with the 0pg/ml standard—two to measure the background absorbance and two for useas the substrate blank wells. The wells were covered with plastic wrapor plate sealer and the samples were incubated at 37° C. for 2 hours.

After the incubation, the plastic wrap or plate sealer was removed, andthe wells were washed using 300 pi per well of plate buffer wash in sixcycles, as supplied and directed in kit. The plate was washed for threecycles, rotated 180°, and washed for three more cycles. Thereafter, 100μl of working conjugate were added to all of the wells, except for thesubstrate blank well which was left empty. The plate was tapped dry on astack of paper towels.

Next, 100 μl of detector antibody (rabbit anti-uPA antiserum in 0.01 MPBS, pH 7.4, a protein stabilizer and 0.1% sodium azide, as supplied inthe uPA ELISA kit, Bayer Diagnostics/Oncogene Science) were added to allof the wells, except for the substrate blank wells. The plates wereincubated at 37° C. for 1.5 hours. During the incubation with thedetector antibody, working conjugate was prepared by diluting theconjugate concentrate (50× goat anti-rabbit IgG horseradish peroxidasein buffer) at 1:50 in conjugate diluent (0.01 M PBS, pH 7.4, BSA and0.01% chloroacetamide), per the Bayer Diagnostics/Oncogene Science uPAELISA kit instructions. The prepared working conjugate was dispensedinto a clean reagent reservoir. Following incubation with detectorantibody, the microtiter plates (or strips) were washed with platewashand tapped dry on a stack of paper towels.

100 μl of the prepared working conjugate as described above were addedto all of the wells, except for the substrate blank wells. The plateswere incubated at room temperature (18-27° C.) for 30 minutes. Duringthe incubation with the working conjugate, the substrate was prepared bydissolving substrate tablets (o-phenylenediamine, OPD, tablets) insubstrate diluent (0.1 M citrate buffer, pH 5.0 and 0.01% H202) byvortexing vigorously. One substrate tablet was dissolved in 4 ml ofsubstrate diluent to make the working substrate. Once prepared, theworking substrate was used within 30 minutes and was not exposed tolight.

After the incubation in working conjugate, the microtiter plates (orstrips of wells) were again washed with platewash solution and theplates were tapped dry on a stack of paper towels. Thereafter, 100 μl ofthe working substrate were added to all of the wells, including thesubstrate blank wells. The microtiter plates were incubated in the darkat room temperature (18-27° C.) for 45 minutes. Stop solution (2.5 NH₂SO₄ solution) was then added to each well to stop the enzymaticreaction. Absorbance was read at 490 nm within 30 minutes.

To perform an overnight assay, diluted samples and standards were addedin duplicate to the specified wells as described for the same-day assay.Thereafter the microtiter plates were covered and incubated at roomtemperature (18-27° C.) for 12-18 hours. On the second day, a 1 hourdetector incubation time was used; the rest of the same-day protocol asdescribed above was used.

To evaluate the results obtained from the Example 1 uPA ELISA, theabsorbance values were averaged for each of the standard and sampledilutions to arrive at the mean absorbance. The concentration of uPA foreach sample was interpolated from the standard curve. A variety ofmicroplate reader software packages are available for analysis ofmicroplate data, e.g., SoftmaxPro™ (Molecular Devices Corporation,Sunnyvale, Calif.; and KC4™, BioTek Instruments, Inc. Winooski, Vt.)that simplify the process. A quadratic curve fitting algorithm (secondorder polynomial) was used. The results for the samples were expressedas pg/ml in the original sample by correcting the value obtained fromthe standard curve by the dilution factor used in the assay, per the kitmanual.

Example 2 Enzyme Linked Immunoassay (ELISA) to Measure Levels of PAI-1in Plasma

Plasma samples were centrifuged in a tabletop microcentrifuge to removeall flocculent material and/or particulate matter. Plasma samples weretypically diluted 1:50 in sample diluent as described below and inaccordance with the instructions accompanying the BayerDiagnostics/Oncogene Science PAI-1 Microtiter ELISA assay. The contentsof the PAI-1 Microtiter ELISA assay manual (Bayer Diagnostics/OncogeneScience, Cambridge, Mass.) are hereby incorporated by reference hereinin their entirety.

The Bayer Diagnostics/Oncogene Science PAI-1 Microtiter ELISA detects0.10 ng/ml of PAI-1 analyte in a test sample. The signal of the 0.10ng/ml standard is greater than two times the zero (background) signal.In addition, the PAI-1 Microtiter ELISA is capable of quantifying PAI-1in the active and latent forms, as well as in complex with uPA or tPA.The assay has been tested for cross-reactivity by challenging withPAI-2, tPA and ovalbumin. No cross-reactivity was detected against theseproteins at high challenge doses.

Detailed Protocol

The PAI-1 Microtiter ELISA kit (commercially available from BayerDiagnostics/Oncogene Science, Cambridge, Mass.) used in this Example hasremovable strips of wells so that the assay can be performed on multipleoccasions, and only the number of wells needed are used. A standardcurve was included each time samples were analyzed. The standard curveperformed for each separate assay requires 12 wells (6 standards run induplicate). The six standards and the test samples were run induplicate. For greater accuracy, each sample was tested at more than oneconcentration.

The PAI-1 standards are housed in six separate vials containing PAI-1.Standards levels were calibrated in nanograms of PAI-1 per ml usinghighly purified PAI-1. As provided, standard 6 contained 1.5 ng/ml ofPAI-1; standard 5 contained PAI-1 at 1.00 ng/ml; standard 4 containedPAI-1 at 0.60 ng/ml; standard 3 contained PAI-1 at 0.30 ng/ml; standard2 contained PAI-1 at 0.10 ng/ml; and standard 1 contained PAI-1 at 0ng/ml.

The appropriate number of 8-well strips was selected from the microtiterplate; the remaining unused strips were saved for subsequent use. Insome cases, the entire plate was used. A working 1× solution ofplatewash buffer was prepared by adding one part platewash concentrateto 19 parts of deionized water and mixing well. The total volumerequired depended on the washing method used, e.g., automatic microplatewasher, manual microplate washer, or hand-held syringe. Approximately 1liter of the wash buffer was needed to prime an automated washer and runone microtiter plate. The platewash buffer was freshly prepared on theday of use and used at room temperature (18-27° C.).

To perform a same-day assay, samples, standards and other kit reagentswere warmed to room temperature prior to addition to the microtiterplate wells and starting the assay. The wells of the microtiter plateswere precoated with monoclonal antibody directed against human PAI-1 ascapture reagent. As necessary, samples were diluted in sample diluentcontaining BSA and 0.1% sodium azide, as provided. (BayerDiagnostics/Oncogene Science PAI-1 ELISA kit instructions). Thestandards and diluted samples were each gently mixed without foaming and100 μl were added in duplicate to the wells of the plates. Four wellswere set up with the 0 ng/ml standard—two to measure the backgroundabsorbance and two for use as the substrate blank wells. The wells werecovered with plastic wrap or plate sealer and the samples and standardswere incubated at room temperature (18-27° C.) for 3 hours.

After the incubation, the plastic wrap or plate sealer was removed, andthe wells were washed using 300 μl per well of plate buffer wash in sixcycles, as supplied and directed in the kit. The plate was washed forthree cycles, rotated 180°, and washed for three more cycles.Thereafter, 100 μl of detector antibody (rabbit anti-PAI-1 antiserum in0.01 M PBS, pH 7.4, a protein stabilizer and 0.1% sodium azide, assupplied in the PAI-1 Microtiter ELISA kit, Bayer Diagnostics/OncogeneScience) were added to all of the wells, except for the substrate blankwells. The plates were incubated at room temperature (18-27° C.) for 1.5hours.

During the incubation with the detector antibody, working conjugate wasprepared 15-30 minutes prior to use by diluting the conjugateconcentrate (50× goat anti-rabbit IgG horseradish peroxidase in buffer)at 1:50 in conjugate diluent (0.01 M PBS, pH 7.4, BSA and 0.01%chloroacetamide), per the Bayer Diagnostics/Oncogene Science PAI-1Microtiter ELISA kit instructions. The prepared working conjugate wasdispensed into a clean reagent reservoir. Following incubation withdetector antibody, the microtiter plates (or strips) were washed withplatewash.

100 μl of the prepared working conjugate as described above were addedto all of the wells, except for the substrate blank wells. The plateswere incubated at room temperature (18-27° C.) for 30 minutes. Duringthe incubation with the working conjugate, the substrate was prepared bydissolving substrate tablets (o-phenylenediamine, OPD, tablets) insubstrate diluent (0.1 M citrate buffer, pH 5.0, and 0.01% H₂0₂) byvortexing vigorously to assure complete solubilization. One substratetablet was dissolved in 2 ml of substrate diluent to make the workingsubstrate. During resuspension, a dark opaque or foil-covered tube wasused to prevent light leakage. Once prepared, the working substrate wasused within 30 minutes and was not exposed to light.

After the incubation in working conjugate, the microtiter plates (orstrips of wells) were again washed with platewash. Thereafter, 100 μl ofthe working substrate were added to all of the wells, including thesubstrate blank wells. The microtiter plates were incubated in the darkat room temperature (18-27° C.) for 45 minutes. Stop solution (2.5NH₂SO₄ solution) was then added to each well to stop the enzymaticreaction. Absorbance was read at 490 nm within 30 minutes. If the platewas not read immediately after the stop solution was added, the platewas kept in the dark at room temperature until the absorbance was read.

To perform an overnight assay, diluted samples and the standards wereadded in duplicate to the specified wells as described for the same-dayassay. Thereafter the microtiter plates were covered and incubated atroom temperature (18-27° C.) for 12-18 hours. On the second day, theremainder of the protocol was followed to develop the plates, asdescribed above for the same-day assay.

To evaluate the results obtained from the Example 2 PAI-1 MicrotiterELISA, the absorbance values were averaged for each of the standard andsample dilutions to arrive at the mean absorbance. Using graph paper,the mean absorbance for each standard was plotted on the y-axis versusthe concentration of PAI-1 (in ng/ml) on the x-axis. The concentrationof PAI-1 was determined for each sample dilution by interpolation fromthe standard curve. A variety of microplate reader software packages areavailable for analysis of microplate data, e.g., SoftmaxPro™ (MolecularDevices Corporation, Sunnyvale, Calif.; and KC4™, BioTek Instruments,Inc. Winooski, Vt.) that simplify the process. A quadratic curve fittingalgorithm (second order polynomial) was used.

Example 3 Enzyme Linked Immunoassay (ELISA) to Measure Levels of theuPA:PAI-1 Complex in Plasma

The Bayer Diagnostics/Oncogene Science Human uPA:PAI-1 ComplexMicrotiter ELISA detects 5 pg/ml of uPA:PAI-1 complex in a test sample.The signal of the 5 pg/ml standard is significantly higher than the zero(background) signal. In addition, the uPA:PAI-1 complex ELISA assay hasbeen tested for specificity against PAI-2, tPA, ovalbumin, free uPA andfree PAI-1 and showed no cross-reactivity against these proteins at highchallenge doses.

Detailed Protocol

The uPA:PAI-1 complex Microtiter ELISA kit (commercially available fromBayer Diagnostics/Oncogene Science, Cambridge, Mass.) used in thisExample has removable strips of wells so that the assay can be performedon multiple occasions, and only the number of wells needed are used. Astandard curve was included each time samples were analyzed. Thestandard curve performed for each separate assay requires 12 wells (6standards run in duplicate). The six standards and the test samples wererun in duplicate. For greater accuracy, each sample was tested at morethan one concentration. The contents of the uPA:PAI-1 complex MicrotiterELISA assay manual (Bayer Diagnostics/Oncogene Science, Cambridge,Mass.) are incorporated by reference herein in their entirety.

The uPA:PAI-1 complex standards are housed in six separate vialscontaining lyophilized uPA:PAI-1. Standards levels were calibrated inpicograms of uPA:PAI-1 complex per ml. The standards were reconstitutedwith 1 ml of high quality deionized water prior to use. As provided,standard 6 contained 200 pg/ml of uPA:PAI-1 complex; standard 5contained uPA:PAI-1 complex at 100 pg/ml; standard 4 contained uPA:PAI-1complex at 40 pg/ml; standard 3 contained uPA:PAI-1 at 10 pg/ml;standard 2 contained uPA:PAI-1 complex at 5 pg/ml; and standard 1contained uPA:PAI-1 complex at 0 pg/ml.

For the uPA:PAI-1 complex assay, plasma samples were diluted at least1:10 prior to application to the microtiter plate. Recommended dilutionsare 1:10, 1:20, or 1:40; intermediate or higher dilutions can be used toobtain multiple values that fall on the standard curve. Serial or otherdilutions were performed in separate tubes and then added directly tothe wells of the microtiter plates. Also according to the BayerDiagnostics/Oncogene Science uPA:PAI-1 complex ELISA manual,p-nitrophenyl p′-guandinobenzoate (NPGB), (0.15 ml of 1000× stock inDMSO) is to be added to plasma samples after collection prior toapplication to the ELISA plate so as to prevent the de novo formation ofuPA:PAI-1 complexes. NPGB is optimally heated at 37° C. for at least 10minutes, with shaking or repeated tapping, in order to resolubilize theDMSO. The NPGB stock was diluted 1:1000 in the sample prior toperformance of the uPA:PAI-1 assay.

The appropriate number of 8-well strips was selected from the microtiterplate; the remaining unused strips were saved for subsequent use. Insome cases, the entire plate was used. A working 1× solution ofplatewash buffer was prepared by adding one part platewash concentrateto 19 parts of deionized water and mixing well. The total volumerequired depended on the washing method used, e.g., automatic microplatewasher, manual microplate washer, or hand-held syringe. Approximately 1liter of the wash buffer was needed to prime an automated washer and runone microtiter plate. The platewash buffer was freshly prepared on theday of use and used at room temperature (18-27° C.).

To perform a same-day assay, samples, standards and other kit reagentswere warmed to room temperature prior to addition to the microtiterplate wells and starting the assay. The microtiter wells were precoatedwith two anti-human uPA monoclonal antibodies. As necessary, plasmasamples were diluted in sample diluent containing BSA and 0.1% sodiumazide, as provided. (Bayer Diagnostics/Oncogene Science uPA:PAI-1complex ELISA kit instructions). The standards and diluted samples wereeach gently mixed (by inverting) without foaming, and 100 μl were addedin duplicate to the wells of the plates. Four wells were set up with the0 pg/ml standard—two to measure the background absorbance and two foruse as the substrate blank wells. The wells were covered with plasticwrap or plate sealer and the samples and standards were incubated at 37°C. for 3 hours.

After the incubation, the plastic wrap or plate sealer was removed, andthe wells were washed using 300 ill per well of plate buffer wash in sixcycles, as supplied and directed in the kit. The plate was washed forthree cycles, rotated 180°, and washed for three more cycles.Thereafter, 100 μl of detector antibody (rabbit anti-PAI-1 antiserum in0.01 M PBS, pH 7.4, a protein stabilizer, 2% normal mouse serum (NMS)and 0.1% sodium azide, as supplied in the uPA:PAI-1 complex MicrotiterELISA kit, Bayer Diagnostics/Oncogene Science) were added to all of thewells, except for the substrate blank wells. The plates were incubatedat 37° C. for 1 hour.

During the incubation with the detector antibody, working conjugate wasprepared by diluting the conjugate concentrate (1000× goat anti-rabbitIgG alkaline phosphatase in buffer) in conjugate diluent (0.01 M PBS, pH7.4, BSA, 2% NMS and 0.01% chloroacetamide), per the BayerDiagnostics/Oncogene Science uPA:PAI-1 complex Microtiter ELISA kitinstructions. Briefly, in a clean reagent reservoir, the conjugateconcentrate was first diluted 1:10 in conjugate diluent to make anintermediate conjugate, and then again diluted 1:100 to make the workingconjugate. The prepared working conjugate was dispensed into a cleanreagent reservoir. Following incubation with detector antibody, themicrotiter plates (or strips) were washed with platewash.

100 μl of the prepared working conjugate as described above were addedto all of the wells, except for the substrate blank wells. The plateswere incubated at 37° C. for 15 minutes. During the incubation with theworking conjugate, the substrate was prepared by reconstituting thesubstrate in substrate diluent, according to the BayerDiagnostics/Oncogene Science uPA:PAI-1 complex ELISA instruction manual.Amplifier solution (provided in the Bayer Diagnostics/Oncogene ScienceuPA:PAI-1 complex ELISA kit) was prepared in the same manner. Both thesubstrate solution and amplifier solution were vortexed vigorously toinsure complete solubilization. Once prepared, the working substrate wasused within 30 minutes and was not exposed to light.

After the incubation in working conjugate, the microtiter plates (orstrips of wells) were again washed with platewash. Thereafter, 50 μl ofthe working substrate were added to all of the wells, including thesubstrate blank wells. The microtiter plates were incubated at roomtemperature (18-27° C.) for 20 minutes. Next, 50 μl of the amplifierwere added to all of the wells, including the substrate blank wells. Themicrotiter plates were incubated at room temperature (18-27° C.) for 15minutes. Stop solution (1 M phosphoric acid solution) was then added toeach well to stop the enzymatic reaction. Absorbance was read at 490 nmwithin 60 minutes. If the plate was not read immediately after the stopsolution was added, the values can deviate from the true readings.

To perform an overnight assay, diluted samples and the standards wereadded in duplicate to the specified wells as described for the same-dayassay. Thereafter the microtiter plates were covered and incubated atroom temperature (18-27° C.) for 12-18 hours. On the second day, theremainder of the protocol was followed to develop the plates, asdescribed above for the same-day assay.

To evaluate the results obtained from the Example 3 uPA:PAI-1 complexMicrotiter ELISA, the absorbance values were averaged for each of thestandard and sample dilutions to arrive at the mean absorbance. Usinggraph paper, the mean absorbance for each standard was plotted on they-axis versus the concentration of uPA:PAI-1 complex (in pg/ml) on thex-axis. The concentration of uPA:PAI-1 complex was determined for eachsample dilution by interpolation from the standard curve. A variety ofmicroplate reader software packages are available for analysis ofmicroplate data, e.g., SoftmaxPro™ (Molecular Devices Corporation,Sunnyvale, Calif.; and KinetiCalc™, BioTek Instruments, Inc. Winooski,Vt.) that simplify the process. A linear or a quadratic curve fittingalgorithm (second order polynomial) was used, depending on whicheveryielded a correlation coefficient closer to 1.0. The results for plasmasamples were expressed as pg/ml in the original sample by correcting thevalue obtained from the standard curve for the dilution factor.

Example 4 Serum uPA Monitoring in Patients with Metastatic Breast Cancer

Pretreatment serum was obtained from 242 metastatic breast cancerpatients enrolled in a double-blind, controlled, phase III trial ofsecond-line hormonal therapy (Fadrozole versus Megace). For serumpreparation, blood was drawn by forearm venipuncture and thencentrifuged at 500×g for 10 minutes at room temperature. The serumsupernatant was collected, aliquotted and stored at −70° C.

Serum uPA levels were determined using the uPA Microtiter ELISA assay(Bayer Diagnostics/Oncogene Science, Cambridge, Mass.) as described inExample 1. A normal cutoff value of 1.75 ng/ml (mean+2 SD) was obtainedfrom the analysis of the sera of 29 healthy women between the ages of40-89. Serum uPA levels above this cutoff value were designated aselevated or increased above normal. For example, since 1.75 ng/ml wasdetermined to be the upper limit of normal according to the assayherein, any higher value, e.g., 1.76, 1.77, 1.8, 2, 2.1 and the like, isconsidered to be an increased or elevated value.

The results of the analysis revealed that 33.5% (81/242) of the patientstested had elevated serum uPA levels compared with the normal serum uPAlevel cutoff in normal individuals. For those patients having anelevated serum uPA level, a trend (p=0.11) toward a decreased responserate (Complete Response (CR)+Partial Response (PR)+Stable Disease) fordisease was determined. In addition, the time to disease progression(UP) was significantly shorter for those patients treated withsecond-line hormone therapy and having elevated serum uPA levelscompared with individuals having normal levels (p=0.013). (FIG. 5).(Table 4).

TABLE 4 Patient response rates as determined by serum uPA status SerumuPA Serum uPA Levels Not Levels Patient Response Elevated ElevatedProgressive Disease 92/161 (57%) 55/81 (68%) Stable + Partial Response69/161 (43%) 26/81 (32%) (PR) + Complete Response (CR) p = 0.11

Overall survival was also significantly shorter for patients withelevated serum uPA levels compared with the serum uPA levels in normalindividuals. (p=0.03). (FIG. 6). These results demonstrate the value ofevaluating patients' serum uPA levels to determine whether the patient'sdisease is progressing toward a more severe or serious stage and todetermine patient outcome. The finding of an increase in serum uPA levelcompared to normal serum uPA level during the course of monitoring apatient having metastatic breast cancer allowed a determination of oneor more of patient treatment efficacy and benefit, UP, and diseaseoutcome.

Example 5 Microtiter Based ELISA to Examine Plasma Levels of PAI-1 andthe Complex of uPA:PAI-1 in Cancer Patient Plasma Samples

Active PAI-1 forms an equimolar covalent complex with active uPA, butnot with inactive pro-uPA (P. A. Andreasen et al., 1986, J. Biol. Chem.,261:7644-7651). Because both enzymes must be in the active form in orderto form complexes, the quantity of uPA:PAI-1 complex found in plasma isdetermined in this example, as well as the quantity of PAI-1 alone. Thedetection and measurement of the uPA:PAI-1 complex may be moreindicative of active proteolysis that is occurring in vivo via theplasminogen activation system than measurement of either the uPA or thePAI-1 analyte alone.

In this example, normal plasma levels for both PAI-1 and uPA:PAI-1complexes were established. In addition, plasma samples from a number ofcancer types were examined to determine the relative plasma levels ofPAI-1 and uPA:PAI-1 complexes in different cancers. The results areparticularly useful in light of the advent of therapies molecularlytargeted to the plasminogen activation system and its components.

The PAI-1 and uPA:PAI-1 complex ELISA assays employed in this Exampleare standardized microtiter-based sandwich ELISAs which utilize anendpoint determination of enzymatic color changes. See, Examples 2 and 3above. The assay run time was approximately six hours for the PAI-1ELISA and five hours for the uPA:PAI-1 complex ELISA.

Plasma samples from healthy individuals were directly collected incollection tubes coated with EDTA as anticoagulant (Becton-Dickinson,Franklin Lakes, N.J.). Red blood cells were centrifuged to pellet,resulting in plasma supernatant, used as a normal human EDTA plasmasample. The normal human EDTA plasma samples were diluted in ELISA kitsample diluent and then analyzed in either the PAI-1 or the uPA:PAI-1complex microtiter ELISA assay as described in Examples 2 and 3,respectively. A standard curve with standards tested in duplicate wasrun in each ELISA. All normal plasma samples were tested in duplicate byat least two different operators. Mean values were obtained for all ofthe samples tested. A total of 80 normal male and 80 normal femaleplasmas were tested in order to determine a normal cutoff. The cutoffwas defined as the mean value +1-two standard deviations.

Cancer patient plasma was then analyzed in both the PAI-1 and uPA:PAI-1complex ELISA assays, as described in Examples 2 and 3 above, withsamples tested in duplicate by at least two different operators. Testsamples were from 50 patients having cancers of the breast, colon, lung,or prostate. Plasma samples from 8 patients having bladder cancer wereevaluated. All other experimental parameters were as described for thenormal plasma samples.

The determination of a normal value was defined as the mean value forthe 160 normal plasma samples +/− two standard deviations. The resultsfrom the cancer plasma samples were compared to the normal range. Any ofthe cancer plasma samples that were above the normal range wereconsidered elevated for either PAI-1 or uPA:PAI-1 complexes, and any ofthe cancer plasma samples that were below the normal range wereconsidered decreased for either PAI-1 or uPA:PAI-1 complexes. Graphicalrepresentations of the results were then generated. (FIG. 2).

Colon Cancer Patient Plasma PAI-1 Levels

A total of 50 plasma samples from colon cancer patients were analyzed inthe PAI-1 microtiter ELISA. Fifteen of the 50 colon cancer plasmas (30%)showed elevated PAI-1 levels when compared with the normal range (63ng/mL). Of the nine stage IV colon cancer plasmas, all nine (100%) hadelevated PAI-1 plasma levels.

Prostate Cancer Patient Plasma PAI-1 Levels

A total of 50 prostate cancer plasma samples were analyzed in the PAI-1microtiter ELISA. Six of the 50 (12%) showed elevated PAI-1 levels whencompared with the normal range (63 ng/mL).

Breast Cancer Patient Plasma PAI-1 Levels

A total of 50 breast cancer plasma samples were analyzed in the PAI-1microtiter ELISA. Twelve of the 50 (24%) showed elevated PAI-1 levelswhen compared with the normal range (63 ng/mL). In stage III breastcancer plasmas, 4/21 (19%) showed PAI-1 levels above the normal range,while in stage IV breast cancer plasmas, 8/29 (28%) showed PAI-1 levelsabove the normal range.

Lung Cancer Patient Plasma PAI-1 Levels

A total of 50 lung cancer plasma samples were analyzed in the PAI-1microtiter ELISA. Twenty of the 50 (40%) showed elevated PAI-1 levelswhen compared to the normal range. In stage II lung cancer plasma 2/10(20%) showed PAI-1 levels above the normal range, in stage III lungcancer plasma 10/32 (31%) showed PAI-1 levels above the normal range andin stage IV lung cancer plasma 5/7 (71%) showed PAI-1 levels above thenormal range.

Colon Cancer Patient Plasma uPA PAI-1 Complex Levels

A total of 49 plasma samples from colon cancer patients were analyzed inthe uPA:PAI-1 complex microtiter ELISA. Eight of the 49 colon cancerplasmas (16%) showed elevated uPA:PAI-1 complex levels when comparedwith the normal range (293 pg/mL). Of the nine stage IV colon cancerplasmas, 6/9 (67%) had elevated uPA:PAI-1 levels. Elevated levels of theuPA:PAI-1 complex compared with normal plasma levels of the uPA:PAI-1complex were also found in plasma from patients having other stages ofcolon cancer as follows: 1/16 (6%) of stage C2 colon cancer plasmas hadelevated levels of the uPA:PAI-1 complex; 1/1 (100%) of stage III coloncancer plasmas had elevated levels of the uPA:PAI-1 complex; nosignificant elevation of uPA:PAI-1 complex levels was observed in stageB1 colon cancer (0/2, 0%); stage B2 colon cancer (0/6, 0%); and stage C1(0/12, 0%); stage D (0/2, 0%).

Prostate Cancer Patient Plasma uPA:PAI-1 Complex Levels

A total of 50 prostate cancer plasma samples were analyzed in theuPA:PAI-1 complex microtiter ELISA. Eight of the 50 (20%) showedelevated uPA:PAI-1 complex levels when compared with the normal range(293 pg/mL).

Breast Cancer Patient Plasma uPA:PAI-1 Complex Levels

A total of 50 breast cancer plasma samples were analyzed in theuPA:PAI-1 complex microtiter ELISA. Twenty-six of the 50 (52%) showedelevated uPA:PAI-1 complex levels when compared with the normal range(293 pg/mL). In stage III breast cancer plasmas, 8/21 (38%) showeduPA:PAI-1 complex levels above the normal range, while in stage IVbreast cancer plasmas, 18/29 (62%) showed uPA:PAI-1 complex levels abovethe normal range.

Lung Cancer Patient Plasma uPA:PAI-1 Complex Levels

A total of 49 lung cancer plasma samples were analyzed in the uPA:PAI-1complex microtiter ELISA. Eight of the 49 (16%) showed elevateduPA:PAI-1 complex levels when compared with the normal range (293 pg/mL)In stage II lung cancer plasma 0/10 (0%) showed uPA:PAI-1 complex levelsabove the normal range; in stage III lung cancer plasma 7/32 (22%)showed uPA:PAI-1 complex levels above the normal range; and in stage IVlung cancer plasma 1/7 (14%) showed uPA:PAI-1 complex levels above thenormal range.

Bladder Cancer Patient Plasma uPA:PAI-1 Complex Levels

For the bladder cancer patient samples analyzed for levels of theuPA:PAI-1 complex in plasma samples, 25% were found to be elevated.

Example 6 Serial Analysis of uPA Levels in Sera from Prostate CancerPatients

Serum samples were obtained from 25 prostate cancer patients. Each ofthe patients had from 4 to 6 serial blood samples drawn, from which theserum component was used. The serum samples were obtained in frozen formand were thawed prior to analysis by ELISA, as described in Example 1,for a determination of the level of uPA over time. The samples wereanalyzed in a serial fashion on a monthly basis, or every two or threemonths over a nine to twelve month period of time. Monitoring uPA levelsin serum samples from patients over time in accordance with the methodsof the present invention provides an advantageous approach to check andexamine the patient's response to cancer therapy or treatment over anextended time period.

It will be appreciated that although, as exemplified here, the serumsamples were frozen, freshly drawn blood samples can be seriallycollected from patients over the desired time intervals, and the freshserum (or plasma) obtained therefrom used equally well for analysis.Table 5 presents representative results of uPA levels determined from 5prostate cancer patients whose serum samples were serially analyzed atsix different time intervals during the monitoring period.

TABLE 5 Patient/ Sample No. Draw Date uPApg/ml 3350-1 April 2000 11763350-2 July 2000 1397 3350-3 August 2000 2537 3350-4 October 2000 18673350-5 November 2000 1317 3350-6 March 2001 1035 3351-1 June 2000 8483351-2 July 2000 1193 3351-3 August 2000 1035 3351-4 September 2000 10073351-5 November 2000 879 3351-6 January 2001 1569 3353-1 December 19991445 3353-2 February 2000 1408 3353-3 April 2000 1322 3353-4 June 20001220 3353-5 August 2000 1405 3353-6 January 2001 701 3360-1 June 20006657 3360-2 September 2000 7727 3360-3 December 2000 1739 3360-4February 2001 1055 3360-5 March 2001 1107 3360-6 April 2001 1896 3362-1March 2000 1535 3362-2 June 2000 1498 3362-3 August 2000 1073 3362-4September 2000 1076 3362-5 November 2000 451 3362-6 February 2001 1017

Example 7 Serum uPA Levels in Patients with Pancreatic Cancer

In this example, uPA levels were determined in sera samples in a phaseIII clinical trial of 188 patients with advanced pancreatic cancer. Asandwich ELISA to detect uPA (Bayer Diagnostics/Oncogene Science,Cambridge, Mass.), as described in Example 1, was employed to determinedthe pretreatment serum uPA levels in 188 pancreatic cancer patients (80females and 108 males) enrolled in a randomized, double-blind,placebo-controlled phase Ill trial. A healthy control group of 47 malesand 62 females was used to determine control serum levels of uPA. Table5 summarizes the results.

TABLE Serum uPA Normal uPA Cutoff Levels Levels (ng/mL) Sample ng/mL(mean ± 2 SD) Normal Males 1.15 1.74 (n = 47) Normal Females (n = 62)1.37 1.94 Normal Females Pre- 1.39 menopausal (n = 47) Normal FemalesPost- 1.31 menopausal (n = 15) Male Pancreatic Cancer 2.00 Patients (n =108) Female Pancreatic 1.80 Cancer Patients (n = 80)

The results showed that serum uPA levels were significantly higher infemale (1.37 ng/ml) compared to male (1.15 ng/ml) healthy controlindividuals (p=0.0002). Within the female control group, there was nosignificant difference in serum uPA levels between pre-menopausal women(1.39 ng/ml, n=47) and post-menopausal women (1.31 ng/ml, n=15),(p=0.39). Cutpoint analysis was performed separately using the mean±2 SDfor female (1.94 ng/ml) and male (1.74 ng/ml) controls. In thepancreatic cancer patients, serum uPA levels were elevated in 28 of 80(35%) of the female patients, and in 53 of 108 (49%) of the malepatients. In addition, the mean serum uPA levels were significantlyhigher in both female (1.80 ng/ml, p<0.00001) and male (2.00 ng/ml,p<0.00001) pancreatic cancer patients when compared to normal controls.These data demonstrate that serum uPA levels are significantly elevatedin both male and female pancreatic cancer patients compared to healthy,gender-matched controls.

Example 8 Serum Levels of the HER-2/neu. CEA and CA19-9 OncoproteinMarkers in Patients with Pancreatic Cancer

In this example, the serum levels of the HER-2/neu, carcinoembryonicantigen (CEA) and CA 19-9 oncoprotein markers were determined in samplestaken from 195 patients with pancreatic cancer using an ELISA assay tospecifically detect the HER-2/neu, CEA and CA 19-9 marker antigens.HER-2/neu levels were quantified using the Bayer Immuno 1® System (BayerCorporation, Tarrytown, N.Y.) in vitro diagnostic assay kit using asandwich ELISA format, as described in Bayer Publication No.DA4-1242M00, Clinical Method, December, 2000 (Product No. T01-4189-51).CEA levels were quantified using the Bayer Immuno 18 System in vitrodiagnostic assay kit using a sandwich ELISA format, as described inBayer Publication No. DA4-1205C99, Clinical Method, March, 1999 (ProductNo. T01-3184-51). CA 19-9 levels were quantified using the Bayer Immuno1® System in vitro diagnostic assay kit using a sandwich ELISA format,as described in Bayer Publication No. DA4-1212K95, Clinical Method,October, 1995 (Product No. T01-3561-51).

The values obtained for these markers in the pancreatic cancer patients'serum are presented in Table 6. For comparison with normal controllevels of the respective markers, the normal cutoff for serum levels ofHER-2/neu is 15 ng/mL, as quantified using the HER-2lneu ELISA assay kit(Bayer Diagnostics/Oncogene Science, Cambridge, Mass.). It is to beunderstood that similar results are obtained using both the BayerDiagnostics/Oncogene Science and the Bayer/Technicon Immuno® Systemsandwich ELISA immunoassay platforms.

The accepted normal range of CEA in the serum of nonsmokers is typically<2.5 to 4.0 ng/mL, and <10 ng/mL in the serum of smokers. (Methods inClinical Chemistry, Eds. A. J. Pesce and L. A. Kaplan, The C.V. MosbyCompany, Washington, D.C., Chpt. on CEA by A. H. Rule, pp. 702-713). Thenormal cutoff in serum for CA 19-9 is typically 37.0 U/mL. (B. C. DelVillano et al., 1983, Clin. Chem., 29:549-552; and R. I. Ritts Jr. etal., 1984, Int. J. Cancer, 33:339-345). An observation of the valueslisted in Table 6 shows that 21/195 (11%) of the pancreatic cancerpatients have elevated serum HER-2/neu levels compared to the normalcontrol value of HER-2/neu in serum; 111/195 (57%) of the pancreaticcancer patients are seen to have elevated CEA levels compared to thenormal control range for nonsmokers, while 71/195 (36%) of thepancreatic cancer patients have elevated CEA levels compared to thenormal control range for smokers; and 162/195 (83%) of the pancreaticcancer patients are seen to have elevated CA 19-9 levels compared to thenormal control cutoff value.

TABLE Serum Levels of the HER-2/neu, CEA and CA 19-9 Oncoprotein MarkersSample HER-2/neu CEA CA 19-9 No. (ng/mL) (ng/mL) U/mL 20045941 10.015.3 >240 20050734 9.81 8.9 >240 20051342 19.18 2.9 206.3 20088733 9.701.3 45.2 20091821 11.13 0.5 >240 20092086 15.97 19.4 >240 201015038.29 >100 >240 20104573 9.95 2.1 17.1 20105380 8.21 4.9 >240 2010551617.50 24.5 >240 20105517 12.49 >100 >240 20110061 9.67 >100 >24020110514 6.88 6.0 >240 20111214 11.07 1.9 >240 20111334 10.04 13.4 >24020111445 11.28 8.5 >240 20111820 17.84 6.3 >240 20111970 6.04 0.6 212.820112105 23.28 49.5 3.1 20114568 10.21 55.9 >240 20114784 10.00 1.8 >24020115323 8.15 >100 >240 20115331 9.45 69.7 >240 20115559 9.74 1.5 131.120115882 12.50 >100 >240 20118172 7.24 42.6 150.9 20118862 15.0913.4 >240 20119233 8.11 6.9 139.4 20119846 12.11 16.1 >240 2012014715.48 2.7 111.0 20121657 8.28 3.3 >240 20121927 19.74 >100 >240 201220299.00 1.5 >240 20125601 12.48 98.7 >240 20126517 12.10 49.7 0.1 201267627.33 20.3 >240 20127099 11.16 6.9 >240 20127404 11.41 >100 >240 2012754815.27 7.9 6.6 20128655 13.55 21.8 >240 20130641 9.44 2.3 >240 2013086221.49 >100 >240 20131082 10.21 0.3 4.9 20131185 11.63 2.1 >240 201315627.01 >100 >240 20132765 13.88 15.5 >240 20133718 8.94 2.8 33.6 2013427913.68 18.3 >240 20134682 8.79 2.6 >240 20135297 23.54 >100 >240 201354569.58 5.2 >240 20136960 18.48 17.7 >240 20137399 11.28 2.2 123.5 2013767210.90 1.6 >240 20140046 12.30 2.3 23.1 20140121 8.35 >100 >240 201401858.87 2.2 >240 20140500 8.04 1.1 5.7 20140845 8.10 4.4 >240 20141310 7.763.0 >240 20142492 13.77 >100 5.4 20144108 10.55 89.5 >240 2014443111.06 >100 >240 20144477 12.10 6.0 >240 20145683 9.45 6.3 >240 2014621811.15 1.9 65.1 20146808 13.58 4.9 35.0 20147388 7.65 6.1 0.2 201473899.81 40.3 >240 20147574 14.24 16.1 >240 20147628 25.56 5.0 >240 2014763011.51 1.7 >240 20147720 7.43 1.6 0.1 20147817 16.38 6.6 >240 2014902211.85 3.5 3.0 20149167 8.54 1.6 >240 20149386 10.33 10.5 >240 2015024512.95 68.6 >240 20150467 7.57 0.8 74.0 20150862 7.17 58.4 >240 2015192810.55 >100 2.0 20152393 9.50 1.8 95.5 20152574 12.38 4.3 >240 201526649.82 1.3 >240 20152671 9.23 2.2 >240 20152674 10.16 5.3 212.4 201533227.56 0.7 >240 20153865 7.39 0.8 >240 20153936 6.07 0.6 64.2 201539708.76 81.6 >240 20155381 12.92 0.5 >240 20155604 14.67 3.7 >240 201560188.12 4.1 >240 20156495 10.96 2.1 2.1 20156809 10.29 4.3 >240 201572427.95 1.9 >240 20157365 16.27 22.9 35.2 20157372 5.72 21.6 >240 201578458.57 41.7 142.8 20158568 9.43 10.9 >240 20158997 10.18 1.5 >240 2015915613.31 1.3 14.7 20161316 7.09 1.8 >240 20161426 8.57 5.3 159.6 201620009.19 40/ >240 20162270 14.15 >100 134.1 20162733 9.34 10.4 >240 201632538.00 0.4 4.2 20163333 7.81 2.0 1.9 20163351 16.09 14.4 >240 2016355410.55 57.5 >240 20163557 7.29 2.1 134.6 20163562 11.61 5.5 >240 2016359410.31 2.8 45.2 20163631 8.37 1.1 223.5 20163671 11.51 0.7 213.5 201640807.65 1.2 0.3 20164102 9.78 53.1 >240 20164443 14.17 88.4 >240 2016444523.57 5.6 >240 20164467 17.27 1.4 >240 20164649 12.30 44.3 >240 2016465116.48 18.8 2.0 20165793 14.58 2.0 25.7 20165804 19.62 0.7 96.0 2016628615.13 9.5 0.9 20166724 10.14 16.6 >240 20167115 12.62 15.7 >240 201672749.45 0.7 10.2 20167318 8.87 0.5 1.4 20167356 9.58 1.6 >240 20167860 9.742.9 >240 20168213 13.80 >100 >240 20168615 8.06 2.1 >240 20168808 10.8255.6 >240 20169306 13.33 1.3 >240 20169306 13.52 1.3 >240 20169451 11.1014.0 >240 20169453 10.68 2.5 >240 20169453 11.36 2.3 >240 20169854 9.8840.9 >240 20169962 8.57 4.7 >240 20170019 13.82 2.1 >240 20170084 5.963.0 >240 20170101 16.86 5.5 >240 20170621 6.13 >100 >240 20170960 9.470.9 1.1 20171007 10.53 9.2 197.7 20171008 11.62 2.7 >240 20171309 6.4511.3 13.3 20171458 10.56 0.9 8.9 20171818 8.31 2.2 >240 2017206512.08 >100 >240 20172090 12.56 3.7 >240 20172225 12.16 26.2 >24020172226 9.16 0.7 >240 20172435 9.01 2.7 >240 20172626 7.30 5.2 >24020172628 10.64 4.7 >240 20172879 10.45 8.2 >240 20173049 9.27 2.3 >24020173212 9.31 5.0 >240 20173648 12.15 81.6 >240 20173676 10.94 19.3 >24020174230 10.42 2.1 155.0 20174434 8.35 3.2 >240 20174435 9.85 2.0 >24020174674 9.90 >100 >240 20175331 9.75 6.3 >240 20175631 7.00 56.5 57.720176100 14.40 >100 >240 20176156 8.91 4.9 215A 20176650 7.23 9.9 11.720176710 11.99 7.5 >240 20176751 6.02 64.1 >240 20176865 9.33 17.2 >24020177134 9.85 0.6 >240 20177232 9.00 2.4 192.8 20177445 7.91 3.7 4.320177637 8.88 1.2 201.8 20178434 9.85 2.2 215.7 20178826 14.63 >100 >24020178953 6.44 1.0 >240 20178962 8.49 1.6 >240 20179496 8.38 1.2 >24020180023 9.50 4.9 20.2 20180795 11.42 2.3 26.2 20181224 13.49 1.4 51.520181962 9.33 6.6 >240 20182177 9.30 10.0 >240 20182554 11.66 >100 >24020182794 14.09 37.3 237.9 20183766 6.51 18.8 9.8 20183902 7.98 2.8 >24020185847 11.05 >100 >240

The contents of all issued and granted patents, patent applications,published PCT and U.S. applications, articles, books, references,reference and instruction manuals, and abstracts as referenced or citedherein are hereby incorporated by reference in their entireties to morefully describe the state of the art to which the invention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

1-44. (canceled)
 45. A method of treating a cancer patient with elevated levels of one or more plasminogen activator system components, comprising: (a) administering an anticancer therapy to a cancer patient identified to have plasminogen activator inhibitor-1 (PAI-1) or a complex of plasminogen activator and plasminogen activator inhibitor-1 (uPA:PAI-1 complex) at a level in the plasma that is above the cutoff value of PAI-1 or the uPA:PAI-1 complex in the plasma of gender-matched normal controls; (b) determining if the level of PAI-1 or the uPA:PAI-1 complex in the plasma of the cancer patient is altered following administration of the anticancer therapy, thereby determining if the treatment regimen should be altered; and (c) administering to the patient an adjusted dosage of the anticancer therapy or a different anticancer therapy if a more aggressive therapy is needed, thereby treating a cancer patient with elevated levels of one or more plasminogen activator system components.
 46. The method of claim 45, wherein the cancer patient has a level of the uPA:PAI-1 complex in the plasma that is above the cutoff value of the uPA:PAI-1 complex in the plasma of gender-matched normal controls.
 47. The method of claim 45, wherein the cancer patient has a level of PAI-1 in the plasma that is above the cutoff value of PAI-1 in the plasma of gender-matched normal controls.
 48. The method of claim 45, wherein the determining step comprises conducting an assay to measure the plasma level of PAI-1 or the uPA:PAI-1 complex prior to and after administering the anticancer therapy and comparing the plasma level of PAI-1 or the uPA:PAI-1 complex prior to administration of the anticancer therapy with the plasma level of PAI-1 or the uPA:PAI-1 complex after administration of the anticancer therapy.
 49. The method of claim 45, wherein the cancer is prostate cancer.
 50. The method of claim 45, wherein the cancer is breast cancer.
 51. The method of claim 45, wherein the cancer is colon cancer.
 52. The method of claim 45, wherein the cancer is lung cancer
 53. The method of claim 45, wherein the anticancer therapy is an anti-plasminogen system therapy.
 54. The method of claim 53, wherein the anti-plasminogen system therapy is a serine protease inhibitor or uPA receptor antagonist.
 55. The method of claim 53, wherein the anti-plasminogen system therapy is a urokinase/plasmin inhibitor.
 56. The method of claim 45, further comprising, prior to step (a), measuring the level of PAI-1 or the uPA:PAI-1 complex in a plasma sample from the cancer patient prior to or at the start of cancer treatment or therapy.
 57. The method of claim 56, wherein the plasma level of PAI-1 or the uPA:PAI-1 complex is determined by an enzyme linked immunosorbent assay.
 58. The method of claim 45, further comprising, prior to step (a), comparing the level of PAI-1 or the uPA:PAI-1 complex in the plasma of the cancer patient to the cutoff value of PAI-1 or the uPA:PAI-1 complex in the plasma of gender-matched normal controls, thereby determining the extent to which the patient's plasminogen activator system components are affected by the cancer.
 59. The method of claim 45, further comprising repeating steps (a)-(c).
 60. The method of claim 45, wherein the dosage of the anticancer therapy is increased if the level of PAI-1 or the uPA:PAI-1 complex increases.
 61. The method of claim 45, wherein the anticancer therapy is changed if the level of PAI-1 or the uPA:PAI-1 complex increases.
 62. The method of claim 45, wherein the dosage of the anticancer therapy is decreased if the level of PAI-1 or the uPA:PAI-1 complex decreases.
 63. A method of treating a cancer patient with elevated levels of one or more plasminogen activator system components, comprising: (a) administering an anticancer therapy to a cancer patient identified to have urokinase plasminogen activator (uPA) at a level in the serum that is above the cutoff value of uPA in the serum of gender-matched normal controls; (b) determining if the level of uPA in the serum of the cancer patient is altered following administration of the anticancer therapy, thereby determining if the treatment regimen should be altered; and (c) administering to the patient an adjusted dosage of the anticancer therapy or a different anticancer therapy if a more aggressive therapy is needed, thereby treating a cancer patient with elevated levels of one or more plasminogen activator system components.
 64. The method of claim 63, wherein the cancer is prostate cancer.
 65. The method of claim 63, wherein the cancer is breast cancer.
 66. The method of claim 63, wherein the cancer is colon cancer.
 67. The method of claim 63, wherein the determining step comprises conducting an assay to measure the serum level of uPA prior to and after administering the anticancer therapy and comparing the serum level of uPA prior to administration of the anticancer therapy with the serum level of uPA after administration of the anticancer therapy.
 68. The method of claim 63, wherein the anticancer therapy is an anti-plasminogen system therapy.
 69. The method of claim 68, wherein the anti-plasminogen system therapy is a serine protease inhibitor or uPA receptor antagonist.
 70. The method of claim 68, wherein the anti-plasminogen system therapy is a urokinase/plasmin inhibitor.
 71. The method of claim 63, further comprising, prior to step (a), measuring the level of uPA in a serum sample from the cancer patient prior to or at the start of cancer treatment or therapy.
 72. The method of claim 71, wherein the serum level of uPA is determined by an enzyme linked immunosorbent assay.
 73. The method of claim 63, further comprising, prior to step (a), comparing the level of uPA in the serum of the cancer patient to the cutoff value of uPA in the serum of gender-matched normal controls, thereby determining the extent to which the patient's plasminogen activator system components are affected by the cancer.
 74. The method of claim 63, further comprising repeating steps (a)-(c).
 75. The method of claim 63, wherein the dosage of the anticancer therapy is increased if the level of uPA increases.
 76. The method of claim 63, wherein the anticancer therapy is changed if the level of uPA increases.
 77. The method of claim 63, wherein the dosage of the anticancer therapy is decreased if the level of uPA decreases.
 78. A method of determining the response of a cancer patient to an anticancer therapy, comprising: (a) administering an anticancer therapy to a cancer patient identified to have plasminogen activator inhibitor-1 (PAI-1) or a complex of plasminogen activator and plasminogen activator inhibitor-1 (uPA:PAI-1) at a level in the plasma that is above the cutoff value of PAI-1 or the uPA:PAI-1 complex in the plasma of gender-matched normal controls; and (b) determining if the level of PAI-1 or the uPA:PAI-1 complex is altered, thereby determining the response of the cancer patient to the anticancer therapy.
 79. The method of claim 78, wherein the cancer is prostate cancer.
 80. The method of claim 78, wherein the cancer is breast cancer.
 81. The method of claim 78, wherein the anticancer therapy is an anti-plasminogen system therapy.
 82. The method of claim 78, wherein the determining step comprises conducting an assay to measure the plasma level of PAI-1 or the uPA:PAI-1 complex prior to and after administering the anticancer therapy and comparing the plasma level of PAI-1 or the uPA:PAI-1 complex prior to administration of the anticancer therapy with the plasma level of PAI-1 or the uPA:PAI-1 complex after administration of the anticancer therapy.
 83. A method of determining the response of a cancer patient to an anticancer therapy, comprising: (a) administering an anticancer therapy to a cancer patient identified to have urokinase plasminogen activator (uPA) at a level in the serum that is above the cutoff value of uPA in the serum of gender-matched normal controls; (b) determining if the level of uPA is altered, thereby determining the response of the cancer patient to the anticancer therapy.
 84. The method of claim 83, wherein the cancer is prostate cancer.
 85. The method of claim 83, wherein the cancer is breast cancer.
 86. The method of claim 83, wherein the anticancer therapy is an anti-plasminogen system therapy.
 87. The method of claim 83, wherein the determining step comprises conducting an assay to measure the serum level of uPA prior to and after administering the anticancer therapy and comparing the serum level of uPA prior to administration of the anticancer therapy with the serum level of uPA after administration of the anticancer therapy. 